Stereocontrolled syntheses of peptide thioesters containing modified seryl residues as probes of antibiotic biosynthesis

Article abstract of DOI:10.1021/jo4007893

Methods have been developed to synthesize tri- and pentapeptide thioesters containing one or more p-(hydroxyphenyl)glycine (pHPG) residues and l-serine, some where the latter is O-phosphorylated, O-acetylated, or exists as a β-lactam. Selection of orthogo

Full text of DOI:10.1021/jo4007893

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pubs.acs.org/joc
Stereocontrolled Syntheses of Peptide Thioesters Containing
Modified Seryl Residues as Probes of Antibiotic Biosynthesis
Nicole M. Gaudelli and Craig A. Townsend*
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: Methods have been developed to synthesize tri-
and pentapeptide thioesters containing one or more p-
(hydroxyphenyl)glycine (pHPG) residues and L-serine, some
where the latter is O-phosphorylated, O-acetylated, or exists as
a β-lactam. Selection of orthogonal protection strategies and
development of conditions to achieve seryl O-phosphorylation
without β-elimination and to maintain stereochemical control,
especially simultaneously at exceptionally base-labile pHPG α-
carbons, are described. Intramolecular closure of a seryl
peptide to a β-lactam-containing peptide and the syntheses of
corresponding thioester analogues are also reported. Mod-
ification of classical Mitsunobu conditions is described in the
synthesis of the β-lactam-containing products, and in a broadly
useful observation, it was found that simple exclusion of light from the P(OEt)3-mediated Mitsunobu ring closure afforded yields
of >95%, presumably owing to reduced photodegradation of the azodicarboxylate used. These sensitive potential substrates and
products will be used in mechanistic studies of the two nonribosomal peptide synthetases NocA and NocB that lie at the heart of
nocardicin biosynthesis, a family of monocyclic β-lactam antibiotics.
INTRODUCTION
■
Four classes of naturally occurring β-lactam antibiotics are
currently known: penicillins, for example, the naturally
occurring penicillin N (1) and the oxidatively related
cephalosporins and cephamycins, clavulanic acid (2) and the
antipodal clavams, carbapenem-3-carboxylic acid (3) and more
highly elaborated carbapenems, e.g., thienamycin, and the
monocyclic β-lactams exemplified by nocardicin A (4) (Figure
1).1 Ironically the last of these families, despite comparative
structural simplicity, stubbornly remains the least understood
biosynthetically. In only the last year the first experimental
advances in a decade have been reported such that now central
questions about how the peptide core of the nocardicins is
constructed can be clearly defined. To rigorously address these
questions we have developed and report here methods to
prepare a series of sensitive peptide thioesters and their
Figure 1. Representative structures of the four classes of naturally
phosphorylated and β-lactam-containing derivatives in stereo-
chemically controlled manner with the aim to serve as
discriminating mechanistic probes of these early biochemical
events.
In 2004, the gene cluster responsible for the biosynthesis of
the nocardicins was isolated and characterized from Nocardia
uniformis ssp. tsuyamanensis.2 Prominent among the encoded
proteins was a pair of nonribosomal peptide synthetases
(NRPSs), NocA and NocB, that together house five complete
modules predicted to synthesize an L,L,D,L,L-pentapeptide and
whose demonstrated amino acid substrates would be expected
to form 5 (Figure 2).3 It is with this discovery, however, that
occurring β-lactam antibiotics.
the key unresolved questions can be posed. First, nocardicin G
(6), the earliest β-lactam-containing precursor of nocardicin A
(4), contains a D,L,D-tripeptide, not a pentapeptide, core, and is
stereochemically inconsistent with derivation from 5.4,5 With
the troublesome identification of L-arginine as the amino acid
specifically activated by module 2 of NocA, it would seem the
Received: April 13, 2013
Published: June 12, 2013
© 2013 American Chemical Society
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Figure 2. Hypothetical biosynthesis of nocardicin A. NocA and NocB together contain five modules which are predicted to produce the
pentapeptide L-pHPG-L-Arg-D-pHPG-L-Ser-L-pHPG (5) which then may go on to form the first β-lactam containing intermediate nocardicin G (6)
and ultimately nocardicin A (4).
initial two N-terminal amino acid residues of 5 are cleaved away
during the biosynthesis of the antibiotic, and the C-terminal
tripeptide arising from modules 3−5 is utilized by way of β-
lactam formation and epimerization at the carboxy terminus to
form nocardicin G (6). The timing and mechanism of
precursor peptide truncation, β-lactam formation, and epime-
rization are not known but can be addressed experimentally by
the synthesis of now definable potential substrates for the
biosynthetic system as set out in the following sections.
To achieve these goals, several synthetic challenges intrinsic
to peptide substrates rich in pHPG residues and serine had to
be addressed. The first was establishing suitable chemical
conditions that prevented racemization/epimerization of the
unusually base-sensitive benzylic α-center of pHPG during
peptide elongation. A second and related challenge entailed the
selection of suitable orthogonal protecting groups that
conformed with limitations that both excluded alkaline
deblocking protocols and resulted in penultimate N-terminally
protected peptides labile to acid deprotection after final thiol
coupling. Third, the seryl hyrdroxymethylene was to be both
activated to behave as a potential electrophile and incorporated
into a β-lactam ring without trivial elimination to dehydroala-
nine. The fourth hurdle was separation and purification of
diastereomers that were unavoidably generated during thioester
formation.
successfully replaced either simply by N-acetylcysteamine
(SNAC) or more accurately by pantetheine to which the
terminal synthetic peptide is attached as its thioester.9,10 Chain
elongation is carried out by condensation (C)-domains, which
catalyze peptide bond synthesis between the upstream PCP-
bound thioester and the downstream PCP-bound amino acid α-
amine (Figure 2). Additionally, other editing domains may be
present in NRPSs including epimerization (E) domains and N-
methylation (M) domains.11 The final module found in these
megasynthetases most often contains a C-terminal thioesterase
(TE)-domain as seen in NocB, which catalyzes the
disconnection of the full-length peptidyl chain from the
adjacent PCP domain.
The modular organization of NocA/B and hypothetical
stepwise assembly of the predicted L,L,D,L,L-pentapeptide 5 is
illustrated in Figure 2. The stereochemistry is predicted on the
basis of the known amino acids activated in each domain12 and
the presence or absence of epimerization domains. In particular,
module 3 contains an epimerase domain (E3), presumably
accounting for the N-terminal D-configuration of nocardicin G,
while module 5 does not.
Of the three chemical events that must occur between the
assembly of pentapeptide 5 and the synthesis of nocardicin G
(6), β-lactam formation and C-terminal epimerization can only
take place after the final pHPG addition in module 5.
Truncation of the pentapeptide to the tripeptide core destined
for nocardicin G (D-pHPG-L-Ser-L-pHPG ultimately arising
from modules 3−5) can logically occur before or after either or
both of the other two reactions. The first synthetic task,
therefore, was the preparation of two sets of peptide scaffolds
varying in C-terminal stereochemistry: protected tripeptides
10a and 10b (D-pHPG-L-Ser-L/D-pHPG) and protected
pentapeptides 16a and 16b (L-pHPG-L-Arg-D-pHPG-L-Ser-L/
D-pHPG, each of which could then be converted to the
corresponding pantetheinyl thioester, appropriately primed for
future enzymatic analysis.
At the outset, it was anticipated that stereochemical control
of the benzylic α-positions of pHPG units would be
problematic based on previous experience with partial and
total syntheses of the nocardicins and vancomycin.4,13,14 The
pKa of the α-hydrogen of a pHPG oxyester is estimated to be
∼23 compared to 26−28 for alanine or serine.15 Thioesters,
RESULTS AND DISCUSSION
■
Synthesis of Potential Tri- and Pentapeptides in
Nocardicin Biosynthesis. Nonribosomal peptide synthatases
are giant modular enzymes that catalyze the assembly of a large
number of bioactive peptides and peptide-derived natural
products using the multiple carrier thiotemplate mechanism.6
In the NRPS paradigm, each canonical module contains at least
a basis set of three domains: an adenylation (A) domain,
peptidyl-carrier protein (PCP), and condensation (C) domain.
The A-domain selectively recognizes and activates, with few
exceptions, an L-α-amino acid in an ATP-dependent reaction
(acyl-adenylation). This mixed anhydride reacts with the S-
terminus of a phosphopantetheine arm, appended by post-
translational modification of the PCP domain, to give rise to
the amino acid-S-PCP thioesters shown in Figure 2.7,8 For
artificial substrate mimics, the S-PCP moiety has been
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however, are of particular interest in the present study, and
substantial depression of the pKa of the α-hydrogen of a pHPG
occurs and is calculated to be ∼20 for pHPG and 25−26 for
alanine or serine. By extension, reductions in pKa can also be
anticipated in active esters generated transiently during peptide
coupling and thioester synthesis. As a consequence, orthogonal
amino acid protections relied on blocking groups that could be
removed by exposure to acid or by hydrogenolysis. Survey of
available peptide coupling reagents demonstrated that benzo-
triazol-1-yl-oxytripyrrolidinophosphonium hexafluorophos-
phate (PyBOP) with the hindered base N,N-diisoproylethyl-
amine (DIEA) was a suitable reagent combination in even the
most polar of solvents including DMF, acetonitrile (ACN), and
DCM and took place without detectible epimerization in good
yields. For the envisioned tri- and pentapeptides, the α-
hydrogen of the pHPG units, whether L- or D-configured, were
always adjacent to either, or both, L-serine and L-arginine and
appeared as a simple doublet in DMSO-d6 owing to vicinal
coupling to the adjacent amide N−H. The chemical shift of this
hydrogen in the D-configuration was reliably diagnostic
appearing upfield compared to the L-diastereomer.
the diastereomeric tripeptides 10a and 10b after selective
removal of blocking groups (Scheme 2). To use the
commercially available N-α-Fmoc-NG-(2,2,4,6,7-pentamethyldi-
hydrobenzofuran-5-sulfonyl)-L-arginine (Fmoc-L-Arg(Pbf)-
OH), it would be necessary to avoid the alkaline Fmoc
deprotection in the presence of any pHPG groups or thioesters.
Accordingly, Fmoc-L-Arg(Pbf)-OH was benzylated to 13,
converted to its corresponding free base by treatment with
20% piperidine in THF, and coupled to Boc-L-pHPG to afford
the differentially protected L,L-dipeptide 15. Compound 10a or
10b was N-deblocked with TFA and joined with 15, after
hydrogenolysis, to give the protected L,L,D,L,L/D-pentapeptides
16a or 16b, respectively, with complete retention of all five
stereocenters.
The benzyl protecting groups were removed from 16a and
16b by catalytic hydrogenation, and the resulting N-Boc/Arg-
Pbf pentapeptides were coupled to pantetheine dimethyl ketal
1116 to give the corresponding protected thioesters. A mild
aqueous workup was employed to wash away byproducts, and
the desired Boc/Pbf-protected peptide thioester was extracted
into organic solvent, concentrated in vacuo, and redissolved in
TFA. The final products 17a and 17b were purified by HPLC
and lyophilized to dryness as their corresponding TFA salts.
The pantetheine thioesters 12a and 12b (Scheme 1) were
prepared analogously. While these tripeptide thioesters could
be separated by HPLC, the pentapeptides 17a and 17b coelute.
The generation of a mixture of diastereomers at the relatively
acidic C-terminal pHPG α-center proved unavoidable in the
syntheses of pantetheinyl and N-acetylcysteaminyl (not shown)
peptide thioesters. The peptide thioesters 12a and 12b could
be individually purified by HPLC as noted above. When placed
in 50 mM phosphate buffer, pH 7.5, the half-life of spontaneous
epimerization at this α-center was estimated by HPLC analysis
to be ∼3 h.
The synthesis of the protected tripeptides 10a and 10b
proceeded from the C- to N-terminus in conventional fashion
using N-Boc and benzyl protecting groups as illustrated in
Scheme 1. The p-toluenesulfonate salt of the bis-benzyl L- or D-
a
Scheme 1. Synthesis of Linear Tripeptide Thioesters
Synthesis of Potential O-Phosphoryl and O-Acetyl
Substrates. The β-lactam ring of nocardicin A is derived from
L-serine in a process where no change in oxidation state occurs
at the seryl β-carbon and clean stereochemical inversion is
observed at this center.17−19 The simplest interpretation of
these experimental observations, presuming the intermediacy of
a peptide precursor, is intramolecular nucleophilic substitution
(SNi) of an activated seryl-hydroxyl in an amide-containing
precursor, possibly through O-phosphorylation or activation by
some other means. Chemical support for this hypothesis came
when phosphorylation of serine was mimicked in vitro by
reaction of a protected serine-containing dipeptide under
Mitsunobu conditions, generating the β-lactam rapidly at room
temperature.4,20 O-Phosphorylation is a particularly attractive
route of seryl activation owing to the ubiquitous utilization of
ATP in NRPS systems and the metabolic intermediacy of O-
phosphoserine.
a
Reaction conditions: (a) Boc-L-Ser, PyBOP, DIEA, DMF, 76%; (b)
In considering the construction of O-phosphoryl tripeptides
18a and 18b (Scheme 3), one immediate concern was ready
elimination under even weakly alkaline conditions to form an
undesired dehydroalanine residue. To avoid this outcome,
installation of a protected O-phosphoryl group was achieved
through the use of phosphoramidite chemistry using tetrazole, a
weak acid, in an approach analogous to oligonucleotide
chemical synthesis.21,22 This transformation was then followed
in the same pot by oxidation of the phosphite to the
corresponding phosphate with a hindered hydroperoxide at
low temperature to suppress potential nucleophilic displace-
(1) TFA, (2) 9, PyBOP, DIEA, DMF, 72%; (c) (1) Pd-OH/C, H2(g),
50 psi, THF, (2) 11, PyBOP, K2CO3, DMF, (3) TFA, 39% over three
steps.
pHPG, 7a and 7b, respectively, was coupled with Boc-L-serine,
N-deprotected by brief TFA treatment, and coupled again to
Boc-D-(p-benzyloxyphenyl)glycine 9 to give the diastereomeric
protected tripeptides 10a or 10b.
The protected pentapeptides 16a and 16b were synthesized
in a convergent approach by combining L,L-dipeptide 15 with
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a
Scheme 2. Synthesis of Pentapeptide Thioesters
a
Reaction conditions: (d) Bn-Br, THF, DIPEA, 40%; (e) (1) 20% piperidine/THF, (2) 13, PyBOP, DIPEA, DCM, 43% over two steps; (f) (1) Pd-
OH/C, H2(g), 50 psi, THF, (2) TFA, 10a or 10b, (3) PyBOP, DIPEA, DCM, 42% over three steps; (g) (1) Pd-OH/C H2(g), 50 psi, THF, (2) 11,
PyBOP, K2CO3, (3) TFA, 35% over three steps.
a
Scheme 3. Synthesis of Activated Tripeptide Thioesters
a
Reaction conditions: (h) (1) di-tert-butyl N,N-diisopropylphosphoramidite, tetrazole, THF, (2) tert-butyl hydroperoxide, 64% over two steps; (i)
(1) Pd-OH/C, H2(g), 50 psi, THF, (2) 11, PyBOP, K2CO3, DMF, (3) TFA, 25% over three steps; (j) acetic anhydride, pyridine, 99%; (k) (1) 11,
PyBOP, K2CO3, DMF, (2) TFA, 39% over two steps.
ment of the freshly generated protected O-phosphate and
reforming the free seryl starting material.
under several conditions due to the presence of the seryl free
phosphate and interference with coupling reagents. This failure
was easily overcome by keeping the phosphate protected as an
acid-labile di-tert-butyl-phosphate, which could be subsequently
deblocked after thiol condensation.
To this end, dibenzyl-DIPA was replaced with di-tert-butyl
N,N-diisopropylphosphoramidite (di-tert-butyl-DIPA).
Although reactions conducted with di-tert-butyl-DIPA and
4,5-dicyanoimidazole repeatedly failed, presumably due to a
suboptimal pKa differential, substitution with tetrazole proved
In practice, the O-phosphoryl functionality was installed in
the protected tripeptide 10a or 10b by treatment with dibenzyl
N,N,-diisopropylphosphoramidite (dibenzyl-DIPA) and 4,5-
dicyanoimidazole (DCI), which was then oxidized with tert-
butyl hydroperoxide to generate the benzyl protected O-
phosphate analogue of 18a or 18b. Although this reaction
sequence met with great success, the final peptide coupling
reaction to produce the desired products 19a and 19b failed
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a
Scheme 4. Synthesis of β-Lactam-Containing Tripeptide Thioesters
a
Reaction conditions: (l) Ox-L-Ser, PyBOP, DIEA, DMF, 64%; (m) DEAD, P(OEt)3, THF 96%; (n) (1) Pd-OH/C, H2(g), 50 psi, THF, (2) HCl,
92% over two steps; (o) (1) 26, 2,6-lutidine, isobutyl chloroformate, cat. N-methylmorpholine, acetone, (2) 3-ANA (25a or 25b), 2,6-lutidine, DMF,
67% over two steps; (p) (1) TFA, (2) Boc2O, DIEA, THF/H2O, 73% over two steps; (q) (1) 11, PyBOP, K2CO3, (2) TFA, 41% over two steps.
to be successful. However, compared to dibenzyl phosphor-
amidite, reactions with di-tert-butyl phosphoramidite suffered
from a longer reaction time, 6 h vs 15 min, and lower yields,
64% vs 78%. This difference most likely owes to the relatively
greater steric bulk of the tert-butyl analogue, its greater
sensitivity to moisture, and the lower purity of the commercial
reagent.
equiv of acetic anhydride in pyridine with protected tripeptide
10a or 10b. The protected O-acetyl tripeptides 20a and 20b
were isolated in quantitative yield with no formation of
diastereomers. The desired thioester substrates 21a and 21b
were obtained by hydrogenolysis of 20a and 20b, respectively,
followed by PyBOP coupling to pantetheine dimethyl ketal 11,
TFA deprotection, and HPLC purification of the separable
diastereomers.
Synthesis of Potential β-Lactam-Containing Thioester
Substrates. The nocardicins share with penicillin N (1)
central NRPSs that synthesize their fundamental peptide
precursors. The tripeptide core of the latter, δ-(L-α-amino-
adipoyl)-L-cysteinyl-D-valine (ACV), is produced by and
released from ACV synthetase. This L,L,D-tripeptide is doubly
cyclized by isopenicillin N synthase to 1.23,24 Hydrolysis of a
three- or five-amino acid peptide is similarly expected from
NocA/B. As noted at the outset, however, the timing of
pentapeptide truncation, C-terminal epimerization, and β-
lactam formation is not known. As a final set of reference
standards and potential substrates to examine these questions,
the last synthetic goal was the preparation of two sets of
pantetheinyl and β-lactam-containing peptides varying in C-
terminal stereochemistry: epi-nocardicin G- and nocardicin G-
pantetheine 29a and 29b in addition to L-pHPG-L-Arg-epi-
nocardicin G- and L-pHPG-L-Arg-nocardicin G-SNAC 31a and
31b.
The desired β-lactam peptidyl thioesters epi-nocardicin G-
pantetheine 29a and nocardicin G-pantetheine 29b were first
constructed as a peptide scaffold in a C- to N-terminal fashion
through the central intermediacy of tert-butyl 3-amino-
cardicinate (epi-3-ANA 25a or 3-ANA 25b) in a manner
analogous to published nocardicin syntheses4 (Scheme 4). To
this end, Sheehan’s 4,5-diphenyl-4-oxazolin-2-one (Ox) N-
protecting group was used as a means of bidentate ligation of
the serine nitrogen, preventing competing aziridine formation
in the downstream Mitsunobu cyclodehydration reaction.25,26
Moreover, dipeptides Ox-L-Ser-L-pHPG and Ox-L-Ser-D-pHPG,
Of practical note, purification of protected phosphoryl
peptides 18a and 18b was conducted in two stages. First,
initial purification was conducted by silica gel chromatography
in which the silica was pretreated overnight with 5%
triethylamine. Chromatographic isolation of di-tert-butylphos-
phoryl tripeptides 18a and 18b without prior silica gel
deactivation resulted in partial deprotection of the di-tert-
butyl-O-phosphoryl blocking groups. This decomposition was
evident from both the low integration of the diastereomeric O-
tert-butyl groups appearing as singlets at ca. 1.3 ppm and 1.2
ppm in 1H NMR spectra and a downfield chemical shift of the
proton- and carbon-decoupled phosphorus singlet from ca. −10
to −6 ppm in 31P NMR spectra acquired in DMSO-d6. Silica
gel purification after triethylamine treatment provided the
1
desired compounds in ∼65% purity according to H NMR
spectroscopy. In a second step, crystallization of either
diastereomer of the partially purified product from ether−
hexanes provided the desired fully protected O-phosphorylated
compounds 18a or 18b as colorless fine needles in >99%
purity. Each was devoid of both the diastereomeric impurity,
1
and the β-phosphate elimination product as established by H
NMR spectroscopic analysis. Compounds 18a and 18b were
then subjected to hydrogenolysis and coupled to pantetheine
dimethyl ketal 11 with PyBOP and K2CO3 in the usual manner,
and the desired thioesters 19a and 19b were obtained after
TFA deprotection and HPLC purification as an inseparable
mixture of C-terminal diastereomers.
In addition to O-phosphorylation, O-acetylation was also
envisioned as a possible means of seryl activation in vivo.
Tripeptides 20a and 20b were generated simply by reacting 1
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23a and 23b, respectively, were prepared from one unit of
either L- or D-tert-butylbenzyloxy-pHPG (22a or 22b) and Ox-
L-Ser in a PyBOP-mediated reaction, which provided the
products in high yield with no pHPG epimerization detectable
the final thioesters as a mixture of C-terminal diastereomers in
an equilibrium ratio of 1:1.1 29a: 29b. The diastereomers were
separable by HPLC purification under acidic conditions.
Substrates 30a and 30b were obtained similarly by substitution
of SNAC for the pantetheine dimethyl ketal 11.
1
by H NMR spectroscopy. Protected β-lactam intermediates
Bearing the planned biochemical experiments in mind, the
intrinsic rate of epimerization at the thioester α-center was
determined by HPLC time-course analysis of purified
nocardicin G-SNAC 30b in 50 mM phosphate buffer at pH
7.5 (Figure 3). The rate of spontaneous epimerization of
24a and 24b were successfully generated from seryl-dipeptides
23a and 23b with retention of stereochemistry at the pHPG α-
center through a modification of the classical Mitsunobu
reaction procedure in which PPh3 was replaced with P(OEt)3; a
reaction explored more fully elsewhere.20,27 Hydrogenolysis of
24a or 24b followed by aqueous acidic extraction and
lyophilization afforded the desired central intermediates epi-3-
ANA·HCl 25a and 3-ANA·HCl 25b, respectively, as white salts.
Of particular note, it was discovered here that excluding light
from the modified Mitsunobu conditions allowed for reaction
yields as high as 96%, an improvement from the previously
reported 84% yield and a cleaner workup. It is suggested that
the omission of light reduces or eliminates DEAD degradation.
In a mechanistic study of the Mitsunobu esterification reaction,
Hughes et al. discovered that in all solvents examined,
formation of the DEAD and PPh3 adduct occurs more rapidly
than either the subsequent alcohol activation to form the
oxyphosphonium intermediate or the ensuing SN2 displace-
ment.28 In the present case where P(OEt)3 is substituted for
PPh3, the formation of the adduct between DEAD and P(OEt)3
may progress at a comparatively slower rate leaving the DEAD
free to photodecompose before it is fully consumed. This factor
could account for lower overall yields and product purity. In
view of these observations, both reaction yields and ease of
product purification for all Mitsunobu-type transformations
may greatly benefit from the exclusion of light.
With the key dipeptide β-lactam-containing intermediates in
hand, syntheses of the protected epi-nocardicin G 27a and
nocardicin G 27b were achieved through the condensation of
Boc-D-pHPG 26 and epi-3-ANA 25a or 3-ANA 25b,
respectively, by way of modified Vaughan conditions for the
generation of mixed anhydrides from isobutylchloroformate.29
Although the reactions to form 27a or 27b involved amide
bond formation of an unhindered carboxylic acid 26 and
primary amines 25a or 25b, modern coupling reagents that
proceed through the generation of an active ester, such as
PyBOP,30 COMU,31 or EDC and DCC,32 all afforded poor
(<10%) to no observable yields of product under standard
conditions. To overcome this unexpected setback, the desired
protected β-lactam products 27a and 27b were prepared
through in situ generation of a mixed anhydride followed by the
addition of 25a or 25b.33 It was found that reactions to produce
protected epi-nocardicin G 27a and protected nocardicin G 27b
from the condensation of 26 and either 25a or 25b required the
addition of N-methylmorpholine. Production of the mixed
anhydride was monitored by the reliably diagnostic precip-
itation of protonated 2,6-lutidine. Conversely, when N-
methylmorpholine is omitted from the reaction, a salt does
not form and amide bond formation is not observed. By
supplementation with N-methylmorpholine according to the
original Vaughan method, the desired products 27a and 27b
were obtained in good yields.
Figure 3. Chemical equilibrium of spontaneous epimerization of
nocardicin G-SNAC to epi-nocardicin G-SNAC. (a) HPLC analysis of
chemical epimerization of the α-hydrogen of the C-terminal pHPG in
nocardicin G-SNAC in 50 mM KiPO4 buffer at pH 7.5 as a function of
time. (b) Extent of chemical epimerization was quantified and plotted
as a one-phase exponential decay. The rate of chemical epimerization
was measured to be 5.5(2) × 10−4 s−1 (k1 + k2).
nocardicin G-SNAC 30b to epi-nocardicin G-SNAC 30a was
measured to be 5.5 0.2 × 10−4 s−1 (k1 + k2) corresponding to
a half-life of ∼21 min. Dynamic equilibrium of the two
diastereomers is achieved in less than 3 h, resulting in a 1.1:1
nocardicin G-SNAC/epi-nocardicin G-SNAC ratio. The slightly
thermodynamically preferred thioester 30b contains a C-
terminal D-pHPG, identical to the biologically observed
nocardicin stereoisomer. By comparison to the pantetheinyl-
tripeptides 12a and 12b, the rate of chemical epimerization of
the β-lactam-containing compounds is ca. 10× faster.
The β-lactam-containing pentapeptides L-pHPG-L-Arg-epi-
nocardicin G-SNAC 31a and L-pHPG-L-Arg-nocardicin G-
SNAC 31b were synthesized in a convergent approach utilized
similarly above for pentapeptides 16a and 16b by combining
the free acid of dipeptide 15 and the free base of either epi-
nocardicin G-SNAC 30b or nocardicin G-SNAC 30 (Scheme
5). Unlike the previous syntheses reported here in which an N-
Boc-protected peptide intermediates were coupled to the
desired thiol as the penultimate step prior to final deprotection,
the synthesis of compounds 31a and 31b required the
With improved syntheses of protected nocardicin G and epi-
nocardicin G established, the desired thioesters epi-nocardicin
G-pantetheine 29a and nocardicin G- pantetheine 29b were
synthesized from either diastereomer of the Boc protected
nocardicin G (28a or 28b) by PyBOP coupling followed by
pantetheine attachment. Acidic deprotection (TFA) produced
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Featured Article
a
Scheme 5. Synthesis of β-Lactam-Containing Pentapeptide Thioesters
a
Reaction conditions: (r) (1) SNAC, PyBOP, DIEA, (2) TFA, 52% over two steps; (s) (1) PyBOP, K2CO3, free acid from hydrogenation of 15, (2)
TFA, 10% over two steps.
formation of the thioester before final amide bond
condensation due to both the absence of a C-terminally
protected carboxylic acid in compounds 28a and 28b and the
restrictions of protecting group orthogonality resulting in an N-
Boc protected compound. Pentapeptide β-lactams 31a and 31b
could be obtained as a mixture of C-terminal diastereomers by
condensation of dipeptide 15 and preinstalled SNAC thioesters
30a or 30b. TFA deprotection produced the desired products
as a separable mixture of diastereomers, which were HPLC
purified under acidic conditions.
such as ramoplanin and Streptomyces calcium-dependent
antibiotics. The tactics and reaction conditions developed
here will be of broader use in biosynthetic investigations of not
only these pHPG-containing natural products but also those
containing phenylglycine and 3,5-dihydroxyphenylglycine res-
idues. Biochemical experiments to address the central questions
of pentapeptide truncation, C-terminal epimerization and β-
lactam formation in the biosynthesis of the nocardicins will be
addressed in due course.
EXPERIMENTAL SECTION
General Experimental Details. H NMR spectra were recorded
■
1
CONCLUSION
■
on a 400 or 600 MHz spectrometer. Proton chemical shifts are
reported in ppm (δ) relative to internal tetramethylsilane (TMS, δ 0.0
ppm) or with the solvent reference relative to TMS (D2O, δ 4.79 ppm,
CDCl3, δ 7.26 ppm, DMSO-d6, 2.50 ppm). Data are reported as
follows: chemical shift [multiplicity (singlet (s), doublet (d), triplet
(t), quartet (q), and multiplet (m)), coupling constants (Hz),
integration]. 13C NMR spectra were recorded on a 400 (101 MHz)
spectrometer with complete proton decoupling. Carbon chemical
shifts are reported in ppm (δ) relative to TMS with the (CD3)2SO (δ
39.52 ppm) or CDCl3 (δ 77.16 ppm) as the internal standard. 31P
NMR spectra were recorded on a 400 (162 MHz) spectrometer with
complete proton and carbon decoupling with no internal standard.
High-resolution mass spectrometry was performed by either fast atom
bombardment (FAB/magnetic sector) or electrospray ionization
(ESI/IT-TOF). All specific rotations were acquired at 589 nm. All
melting points were uncorrected. All purchased chemical were used as
received.
Syntheses of tri- and pentapeptides incorporating seryl, O-
phosphoseryl, and β-lactam moieties have been developed to
study monocyclic β-lactam antibiotic biosynthesis. The O-
phosphoryl modification was achieved without competing
formation of dehydroalanyl β-elimination products, and four-
membered ring closures of seryl residues to the β-lactam could
be carried out without accompanying epimerization in a
modified Mitsunobu reaction where P(OEt)3 replaced conven-
tionally used PPh3.
During this phase of the work, it was observed that
protection of the reaction from ambient light by wrapping
the reaction flask in aluminum foil raised the yield of the
Mitsunobu ring formation to >95% and simplified product
isolation. We attribute the increased efficiency of this reaction
to reduced photodecomposition of the DEAD reagent.
Unexpected difficulty in peptide bond formation to the β-
lactam α-amine could be overcome by resorting to classical
mixed anhydride-mediated coupling, but only in the presence of
catalytic N-methylmorpholine.
Through careful choice of appropriately orthogonal protect-
ing groups, the absolute configuration of all α-centers was
preserved, notably the especially acidic pHPG units, to
assemble the tri- and pentapeptides 10a, 10b and 16a, 16b,
respectively, with complete stereochemical control. Conversion
of the C-terminal pHPG residue, however, to SNAC and
pantetheine thioesters gave epimerization at the α-carbon that
proved unavoidable, but for several of the final products,
separation by HPLC was possible. Still, in aqueous buffer at
room temperature, spontaneous epimerization occurred slowly
in the peptide products, but ca. 10-fold more rapidly in the β-
lactam thioesters with a half-life of ca. 20 min.
Preparative and analytical HPLC purifications were analyzed via a
multiwavelength UV−vis detector in conjunction with a reversed-
phase C18(2) preparatory column (250 × 21.20 mm ID). Mobile-
phase conditions included one of the following: Prep method A (water
+ acetonitrile (ACN) +0.1% TFA): 0−5 min isocratic 13% water 87%
ACN + 0.1% TFA, 5−25 min gradient 13% to 50% ACN + 0.1% TFA,
25−30 min 50% ACN to 13% ACN, 30−35 min isocratic 13% water
87% ACN + 0.1% TFA. Flow rate = 6.5 mL/min. Prep method B
(water + ACN + 0.1% TFA): 0−25 min gradient 15−80% ACN +
0.1% TFA, 25−30 min 80% to 15% ACN + 0.1% TFA, 30−35 min
15% ACN + 85% water +0.1% TFA. Flow rate =6.5 mL/min.
Analytical HPLC purifications were analyzed with a multi-
wavelength UV−vis detector in conjunction with a reverse phase
phenyl hexyl analytical column (250 × 4.60 mm i.d.). Analytical
method A (water + ACN + 0.1% TFA): 0−5 min isocratic 93% water
+7% ACN +0.1% TFA, 5−22 min gradient 7% to 50% ACN + 0.1%
TFA, 22−25 min gradient 50% to 7% ACN + 0.1% TFA, 25−35 min
isocratic 93% water +7% ACN + 0.1% TFA. Flow rate = 1.0 mL/min.
Analytical method B (water + ACN + 0.1% TFA): 0−20 gradient 7%
to 50% ACN + 0.1% TFA, 20−25 min gradient 50% to 7% ACN +
0.1% TFA, 25−35 min isocratic 93% water +7% ACN + 0.1% TFA.
Flow rate = 1.0 mL/min.
The nonproteinogenic amino acid pHPG appears often in
NRPS-derived natural products apart from the nocardicins,
notably the glycopeptides vancomycin, A47934, chloroeremo-
mycin, complestatin, as well as other antimicrobial compounds
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Featured Article
Benzyl-L-[p-(benzyloxy)phenyl]glycine Toluenesulfonate (7a). To
a 500 mL round-bottomed flask, equipped with a magnetic stir bar, L-
[p-(benzyloxy)phenyl]glycine14 (10.0 g, 38.9 mmol) was suspended in
reagent-grade benzyl alcohol (100 mL) and benzene (140 mL) p-
toluenesulfonic acid monohydrate (8.87 g, 46.7 mmol) warmed to
reflux under Dean−Stark conditions for 12 h. The solution was cooled
to room temperature and added directly to 500 mL of Et2O. The
precipitate was filtered and resuspended in 1 L of Et2O and vigorously
stirred for 1 h to remove residual benzyl alcohol. The precipitate was
69.7, 66.7, 62.5, 56.9, 56.2, 28.6. HRMS (FAB): calcd for C30H35N2O7
535.2443, found 535.24288 [M + H]+.
N-tert-Butyloxycarbonyl-D-p-(benzyloxy)phenyl]glycine (9). In a
500 mL Erlenmeyer flask equipped with a magnetic stir bar was
dissolved D-[p-(benzyloxy)phenyl]glycine (10.00 g, 38.87 mmol) in
250 mL of 0.5 M NaOH (aq) and to this solution was added di-tert-
butyl dicarbonate (9.33 g, 42.75 mmol) in 300 mL of reagent-grade
THF. The reaction was stirred at room temperature for 12 h. The
mixture was transferred to a 1-L separatory flask, and the THF was
partitioned from the aqueous fraction with 200 mL of Et2O and
removed. The aqueous layer was transferred to a 1-L Erlenmeyer flask,
cooled to 0 °C in an ice bath, and acidified to pH 2.0 with concd HCl.
The acidified aqueous mixture was extracted with EtOAc (3 × 100
mL), and the organic extractions were pooled and washed with brine
filtered to afford the product as a white salt (19.46 g, 96%). [α]26
=
D
1
26.3 (c = 1.0, MeOH). H NMR (400 MHz; DMSO-d6): δ 8.88 (s,
3H), 7.54 (d, J = 8.1 Hz, 2H), 7.47−7.23 (m, 12H), 7.14 (d, J = 7.9
Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 5.31 (s, 1H), 5.24, 5.19 (ABq, JAB
=
12.4 Hz, 2H), 5.15 (s, 2H), 2.29 (s, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 168.5, 159.2, 145.3, 138.0, 136.8, 135.1, 129.8, 128.5,
128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 126.6, 125.6, 124.5,
115.3, 69.3, 67.2, 63.0, 55.0, 20.9. HRMS (FAB): calcd for C22H22NO3
348.15997, found 348.15966 [M + H]+.
(1 × 75 mL) and concentrated in vacuo to afford the product as a
1
white foam (10.28 g, 74%). [α]25 = −93.3 (c = 1.0, MeOH). H
D
NMR (400 MHz; DMSO-d6): δ 7.44−7.30 (m, 8H), 6.96 (d, J = 8.8
Hz, 2H), 5.08 (s, 2H), 5.02 (d, J = 8.0 Hz, 1H), 1.38 (s, 9H). 13C
NMR (101 MHz, DMSO-d6): δ 172.6, 157.9, 155.1, 137.1, 130.3,
128.9, 128.5, 127.8, 127.6, 114.5, 78.2, 69.2, 57.3, 28.2. HRMS (FAB):
calcd for C20H24NO5 358.16545, found 358.16500 [M + H]+.
N-tert-Butyloxycarbonyl-D-[p-(benzyloxy)phenyl]glycine-L-serine-
L-[p-(benzyloxy)phenyl]glycine Benzyl Ester (10a). To a 250 mL
round-bottomed flask equipped with a magnetic stir bar, 8a (7.87 g,
14.72 mmol) was dissolved in 100 mL of a 1:3 DCM/TFA solution
and stirred at room temperature for 30 min. The solution was
concentrated in vacuo, and residual TFA was removed by azeotropic
distillation with toluene (2 × 75 mL) and placed under high vacuum
for 20 min.
Benzyl-D-[p-(benzyloxy)phenyl]glycine Toluenesulfonate (7b).
The title compound was prepared and purified analogously to
compound 7a by replacing L-[p-(benzyloxy)phenyl]glycine with D-
[p-(benzyloxy)phenyl]glycine (10.0 g, 38.9 mmol). The product was
obtained as a white salt (19.46 g, 96%). [α]23 = −116.3 (c = 1.0,
D
1
EtOAc). [α]26 = −21.3 (c = 1.0, MeOH). H NMR (400 MHz;
D
DMSO-d6): δ 8.88 (br s, 3H), 7.54 (d, J = 8.1 Hz, 2H), 7.47−7.23 (m,
12H), 7.14 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H), 5.31 (s, 1H),
5.24, 5.19 (ABq, JAB = 12.4 Hz, 2H), 5.15 (s, 2H), 2.29 (s, 3H). 13C
NMR (101 MHz, DMSO-d6): δ 169.0, 159.6, 145.9, 138.3, 137.2,
135.6, 130.2, 129.0, 128.9, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2,
126.9, 126.0, 125.0, 116.0, 69.7, 67.7, 63.4, 55.4, 21.3. HRMS (FAB):
calcd for C22H22NO3 348.15997, found 348.15923 [M + H]+.
In a separate 250 mL round-bottomed flask equipped with a
magnetic stir bar, 9 (5.26 g, 14.72 mmol) and DIEA (2.56 mL, 14.72
mmol) were dissolved in 30 mL of reagent-grade DMF and cooled to
0 °C in an ice bath. The freshly deprotected 8a was separately
dissolved in 30 mL of reagent-grade DMF, DIEA (5.12 mL, 29.44
mmol) was added, and the solution was cooled to 0 °C in an ice bath.
When both solutions were sufficiently cooled, PyBOP (11.16 g, 21.49
mmol) was added to the flask containing 9. After 1 min, the solution
containing 8a was added dropwise over 2 min to the activated
carboxylic acid and the reaction was allowed to stir 0 °C to room
temperature for 3 h. The reaction was diluted with 200 mL of EtOAc
and washed with satd aq NH4Cl (2 × 75 mL), satd aq NaHCO3 (2 ×
75 mL), and brine (1 × 75 mL). The organic layer was concentrated in
vacuo, and the product was purified by silica gel chromatography with
a gradient of 60:40 EtOAc/Hex to 70:30 EtOAc/Hex over 3 L to
afford the product as a white foam (8.00g, 70%). [α]25D = 0.2 (c = 1.0,
EtOAc). 1H NMR (400 MHz; DMSO-d6): δ 8.64 (d, J = 6.4 Hz, 1H),
8.24 (d, J = 7.6 Hz, 1H), 7.44−7.23 (m, 20H), 7.01 (d, J = 8.8 Hz,
2H), 6.94 (d, J = 8.8 Hz, 2H), 5.42 (d, J = 6.8 Hz, 1H), 5.22 (d, J = 8.4
Hz, 1H), 5.11 (s, 4H), 5.07 (s, 2H), 4.87 (t, J = 5.3 Hz, 1H), 4.41 (br
q, J = 6.6 Hz, 1H), 3.55 (br t, J = 5.3 Hz, 2H), 1.36 (s, 9H). 13C NMR
(101 MHz, DMSO-d6): δ 170.4, 170.3, 169.9, 158.3, 157.7, 154.8,
154.8, 137.1, 137.0, 135.8, 131.2, 129.1, 128.5, 128.5, 128.4, 128.3,
128.0, 128.0, 127.9, 127.8, 127.6, 127.6, 127.5, 114.9, 114.4, 78.4, 69.2,
69.2, 66.1, 61.6, 57.1, 55.9, 54.8, 28.2. HRMS (FAB): calcd for
C45H48N3O9 774.33906, found 774.33708 [M + H]+.
N-tert-Butyloxycarbonyl-L-serine-L-[p-(benzyloxy)phenyl]glycine
Benzyl Ester (8a). In a 250 mL round-bottomed flask equipped with a
magnetic stir bar, Boc-L-serine (4.0 g, 19.49 mmol) and DIEA (2.52
mL, 19.49 mmol) were dissolved in 30 mL of freshly distilled DCM
and 10 mL of reagent-grade DMF and cooled to 0 °C in an ice bath. In
a separate flask, 7a (10.12 g, 19.49 mmol) was dissolved in 30 mL of
reagent-grade DMF, to this suspension was added DIEA (5.03 mL,
38.98 mmol). and the mixture was cooled to 0 °C with an ice bath.
When both solutions had come to temperature, PyBOP (11.16 g,
21.49 mmol) was added to the flask containing Boc-L-Ser. After 1 min,
the amine solution containing 7a was added dropwise over 2 min to
the activated carboxylic acid and the reaction mixture was allowed to
stir at 0 °C to room temperature for 3 h. The solution was diluted with
200 mL of EtOAc and washed with satd aq NH4Cl (2 × 75 mL), satd
aq NaHCO3 (2 × 75 mL), and brine (1 × 75 mL). The organic layer
was concentrated in vacuo and purified by silica gel chromatography
with a gradient of 40:60 EtOAc/Hex to 50:50 EtOAc/Hex over 3 L to
obtain the product as a white foam (7.87g, 76%). [α]22 = 42.8 (c =
D
1
1.0, EtOAc). H NMR (400 MHz; DMSO-d6): δ 8.55 (d, J = 6.8 Hz,
1H), 7.46−7.21 (m, 12H), 7.00 (d, J = 8.8 Hz, 2H), 6.75 (d, J = 8.4
Hz, 1H), 5.41 (d, J = 6.8 Hz, 1H), 5.13, 5.10 (ABq, JAB = 12.8 Hz, 2H),
5.12 (s, 2H), 4.80 (t, J = 5.8 Hz, 1H), 4.11 (q, J = 6.2 Hz, 1H), 3.61−
3.56 (m, 1H), 3.53−3.47 (m, 1H), 1.37 (s, 9H). 13C NMR (101 MHz,
DMSO-d6): δ 170.4, 170.3, 158.3, 155.2, 137.0, 136.7, 129.0, 128.4,
128.3, 128.2, 128.0, 127.8, 127.6, 127.5, 114.9, 78.2, 69.2, 66.2, 61.9,
56.6, 55.7, 28.2. HRMS (FAB): calcd for C30H35N2O7 535.2443, found
535.24361 [M + H]+.
N-tert-Butyloxycarbonyl-L-serine-D-[p-(benzyloxy)phenyl]glycine
Benzyl Ester (8b). The title compound was prepared and purified
analogously to compound 8a by replacing 7a with 7b (10.0 g, 49.4
mmol). The product was obtained as a white foam (7.87g, 76%).
[α]22D = −43.2 (c = 1.0, EtOAc). 1H NMR (400 MHz; DMSO-d6): δ
8.57 (d, J = 7.2 Hz, 1H), 7.47−7.23 (m, 12H), 7.01 (d, J = 8.8 Hz,
2H), 6.68 (d, J = 8.4 Hz, 1H), 5.44 (d, J = 7.2 Hz, 1H), 5.14, 5.12
(ABq, JAB = 12.7 Hz, 2H), 5.12 (s, 2H), 4.87 (br t, J = 5.8 Hz, 1H),
4.18 (br q, J = 6.9 Hz, 1H), 3.56 (t, J = 5.6 Hz, 2H), 1.40 (s, 9H). 13C
NMR (101 MHz, DMSO-d6): δ 170.9, 170.8, 158.7, 155.6, 137.5,
136.2, 129.3, 128.9,128.8, 128.7, 128.4, 128.3, 128.1, 128.0, 115.4, 78.7,
N-tert-Butyloxycarbonyl-D-[p-(benzyloxy)phenyl]glycine-L-serine-
D-[p-(benzyloxy)phenyl]glycine Benzyl Ester (10b). The title
compound was prepared and purified analogously to compound 10a
by replacing 8a with 8b (7.87 g, 14.72 mmol). The product was
obtained as a white foam (8.23 g, 72%). [α]25 = −62.0 (c = 1.0,
D
EtOAc). 1H NMR (400 MHz; DMSO-d6): δ 8.66 (d, J = 6.9 Hz, 1H),
8.23 (d, J = 7.8 Hz, 1H), 7.46−7.23 (m, 20H), 7.01 (d, J = 8.8 Hz,
2H), 6.93 (d, J = 8.8 Hz, 2H), 5.45 (d, J = 7.1 Hz, 1H), 5.24 (d, J = 8.1
Hz, 1H), 5.16, 5.13 (ABq, JAB = 12.5 Hz, 2H), 5.12 (s, 2H), 5.08 (s,
2H), 4.90 (t, J = 5.2 Hz, 1H), 4.44 (br q, J = 6.1 Hz, 1H), 3.56−3.44
(m, J = 5.2 Hz, 2H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ
170.8, 170.4, 158.7, 158.2, 137.55, 137.5, 136.2, 131.5, 129.4, 129.0,
128.92, 128.89, 128.83, 128.79, 128.6, 128.4, 128.32, 128.28, 128.1,
128.0, 115.3, 114.9, 78.9, 69.69, 69.65, 66.6, 62.2, 56.3, 55.2, 28.6.
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The Journal of Organic Chemistry
Featured Article
HRMS (FAB): calcd for C45H48N3O9 774.33906, found 774.33786 [M
+ H]+.
(FAB): calcd for C30H42N5O10S 664.26524, found 664.26512 [M +
H]+.
D-(p-Hydroxyphenyl)glycine-L-serine-D-(p-hydroxyphenyl)-
glycylpantetheine (12b). The title compound was prepared and
purified analogously to compound 12a by replacing 10a with 10b (580
mg, 0.75 mmol). The product was obtained as the TFA salt (229 mg,
39%). Isolation of 2 mg of diasteromerically pure material was
Pantetheine Dimethyl Ketal (11). To a 500 mL round-bottomed
flask, equipped with a magnetic stir bar, D-pantothenic acid
hemicalcium salt (5.00 g, 10.49 mmol), p-toluenesulfonic acid
monohydrate (4.79 g, 25.18 mmol), and 5.00 g of 3 Å molecular
sieves were suspended in 250 mL of reagent grade acetone. The flask
was capped, and the suspension was stirred at room temperature for
12 h. The thick slurry was filtered through Celite and washed with 3 ×
100 mL of acetone, and the filtrate was concentrated to a viscous oil.
The oil was dissolved in 200 mL of EtOAc, washed with brine (2 ×
100 mL), and further dried with Na2SO4. The EtOAc was removed in
vacuo, and just before the EtOAc was fully removed, hexane was added
slowly to precipitate a solid that was dried under high vacuum and
used in the next step without further purification.
To a 250 mL round-bottomed flask equipped with a magnetic stir
bar, freshly prepared D-pantothenic dimethyl ketal (3.69 g, 14.23
mmol) was dissolved in 90 mL of freshly distilled THF, and treated
with 1′,1′-carbonyldiimidazole (3.46 g, 21.34 mmol) and stirred for 1
h at room temperature. To this solution was added cysteamine
hydrochloride (2.42 g, 21.34 mmol) and stirred at room temperature
for 12 h. The THF was concentrated in vacuo, and 100 mL of DCM
was added. The organic solution was washed satd aq NH4Cl (1 × 50
mL) and brine (1 × 50 mL), dried with Na2SO4 filtered, and
concentrated to an oil. The oil was purified by silica gel flash
chromatography with a mobile phase of 70:30 EtOAc/Hex to 100%
EtOAc to produce the desired product as a white solid (2.71 g, 81%).
[α]23D = 33.4 (c = 1.0, MeOH). 1H NMR (400 MHz; CDCl3): δ 7.01
(t, J = 5.8 Hz, 1H), 6.62 (t, J = 5.1 Hz, 1H), 4.03 (s, 1H), 3.64 (d, J =
11.6 Hz, 1H), 3.51 (qd, J = 12.3, 6.4 Hz, 2H), 3.38 (qd, J = 13.5, 6.4
Hz, 2H), 3.23 (d, J = 11.7 Hz, 1H), 2.61 (dtd, J = 8.4, 6.6, 1.8 Hz, 2H),
2.43 (t, J = 6.2 Hz, 2H), 1.41 (s, 3H), 1.38 (s, 3H), 1.36 (t, J = 8.5 Hz,
1H), 0.98 (s, 3H), 0.92 (s, 3H). 13C NMR (101 MHz, CDCl3): δ
171.2, 170.30, 99.1, 77.2, 71.4, 42.5, 36.1, 35.0, 33.0, 29.5, 24.5, 22.2,
18.9, 18.6. HRMS (FAB): calcd for C14H27N2O4S 319.1692, found
319.16924 [M + H]+.
1
achieved using analytical method A. H NMR (400 MHz; D2O): δ
7.29 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz,
2H), 6.84 (d, J = 8.7 Hz, 2H), 5.46 (s, 1H), 5.07 (s, 1H), 4.48 (t, J =
5.6 Hz, 1H), 3.87 (s, 1H), 3.63 (d, J = 5.6 Hz, 2H), 3.40 (d, J = 11.2
Hz, 1H), 3.30−3.26 (m, 5H), 3.04−2.97 (m, 2H), 2.19 (app td, J =
6.5, 2.1 Hz, 2H), 0.81 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz,
D2O): δ 201.0, 175.0, 173.9, 171.2, 169.0, 157.2, 156.4, 129.8, 129.7,
126.3, 123.5, 123.5, 116.3, 116.0, 75.7, 68.4, 63.6, 60.8, 56.1, 55.4, 38.6,
38.3, 35.1, 28.1, 20.5, 19.1. HRMS (FAB): calcd for C30H42N5O10S
664.26524, found 664.26484 [M + H]+.
N-α-Fmoc-NG-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl(Pbf))-L-arginine Benzyl Ester (13). To a 500 mL round-
bottomed flask equipped with a magnetic stir bar was dissolved Fmoc-
L-Arg(Pbf)-OH (20.0 g, 30.83 mmol) in 100 mL of freshly distilled
DCM. To this solution were added DIEA (6.44 mL, 37.00 mmol),
benzyl bromide (4.39 mL, 36.99 mmol), and a catalytic amount of
DMAP (376 mg, 3.08 mmol). The reaction mixture was stirred at
room temperature for 12 h. The solution was concentrated in vacuo,
and the crude product was directly purified by silica gel
chromatography with 80:20 DCM/acetone to afford the product as
1
a white foam (4.47 g, 20%). [α]25 = −0.68 (c = 1.0, EtOAc). H
D
NMR (400 MHz; DMSO-d6): δ 7.89 (d, J = 7.5 Hz, 2H), 7.85−7.83
(m, 1H), 7.71 (d, J = 7.5 Hz, 2H), 7.43−7.40 (m, 2H), 7.35−7.30 (m,
6H), 6.69 (br s, 1H), 6.45 (br s, 1H), 5.10 (s, 2H), 4.34−4.27 (m,
2H), 4.22 (t, J = 7.0 Hz, 1H), 4.08−4.04 (m, 1H), 3.36 (s, 1H), 3.06−
3.02 (m, 2H), 2.91 (s, 2H), 2.50 (s, 3H), 2.44 (s, 3H), 1.99 (s, 3H),
1.73 (br s, 1H), 1.63 (br s, 1H), 1.45 (br s, 2H), 1.38 (s, 3H), 1.38 (s,
3H). 13C NMR (101 MHz, DMSO-d6): δ 172.1, 157.4, 156.1, 143.8,
143.7, 140.7, 137.3, 135.9, 131.4, 128.9, 128.4, 128.0, 127.7, 127.3,
127.1, 125.2, 125.2, 124.3, 121.4, 120.1, 120.0, 116.3, 109.7, 86.3, 65.9,
65.7, 53.8, 46.6, 42.5, 28.3, 19.0, 17.6, 12.3. HRMS (FAB): calcd for
C41H47N4O7S 739.31655, found 739.31607 [M + H]+.
D-(p-Hydroxyphenyl)glycine-L-serine-L-(p-hydroxyphenyl)-
glycylpantetheine (12a). In a 250 mL pressure flask was dissolved
protected tripeptide 10a (580 mg, 0.75 mmol) in 20 mL of reagent
grade THF, and to this solution was added a catalytic amount of Pd-
OH/C. The mixture was vigorously shaken under 50 psi of H2
overnight. The mixture was filtered through Celite, which was washed
with 200 mL of THF and concentrated in vacuo to a white foam and
used in the next step without further purification.
tert-Butyl-L-(p-hydroxyphenyl)glycine (14). In a 1 L Erlenmeyer
flask equipped with a magnetic stir bar was dissolved L-(p-
hydroxyphenyl)glycine (20.00 g, 119.57 mmol) in 300 mL of 1.0 M
NaOH, and to this solution was added di-tert-butyl dicarbonate (31.31
g, 143.48 mmol) in 500 mL of reagent-grade THF. The reaction
mixture was stirred at room temperature for 12 h. The mixture was
transferred to a 1 L separatory funnel, and the THF was partitioned
with 200 mL of Et2O and removed. The aqueous layer was transferred
to a 1 L Erlenmeyer flask, cooled to 0 °C with an ice bath, and
acidified to pH 2.0 with concd HCl. The acidified aqueous mixture was
extracted with EtOAc (3 × 100 mL), and the organic extractions were
pooled, washed with brine (1 × 75 mL), and concentrated in vacuo to
a viscous oil. The product was crystallized as a white solid from
In a 25 mL round-bottomed flask equipped with a magnetic stir bar
was dissolved the freshly deprotected tripeptide (380 mg, 0.75 mmol)
in 5 mL of reagent-grade DMF. To this solution were added K2CO3
(291 mg, 2.27 mmol) and PyBOP (471 mg, 0.91 mmol) followed by
11, and the reaction mixture was stirred at room temperature for 1 h.
The solution was diluted with 35 mL of EtOAc, washed with satd aq
NH4Cl (2 × 15 mL) and satd aq NaHCO3 (1 × 15 mL), and
concentrated in vacuo. The residue was redissolved in 2.0 mL of 1:1
ACN/H2O and purified according to prep method B as a mixture of
diastereomers. The product was collected, frozen on dry ice, and
lyophilized to dryness. The lyophilized powder was dissolved in TFA
for 10 min, concentrated in vacuo, redissolved in 2 mL of 80:20 H2O/
ACN with 0.1% TFA, and purified with prep method B. The product
was collected, frozen on dry ice and lyophilized to dryness to afford the
product as the white TFA salt (205 mg, 35%). Isolation of 2 mg of
diasteromerically pure material was achieved using analytical method
A. 1H NMR (400 MHz; D2O): δ 7.24 (d, J = 8.8 Hz, 2H), 7.23 (d, J =
8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.46 (s,
1H), 5.04 (s, 1H), 4.42 (X of ABX, J = 6.4, 4.8 Hz, 1H), 3.86 (s, 1H),
3.74 (AB of ABX, J = 11.6, 6.4, 4.8 Hz, 2H), 3.39 (d, J = 11.2 Hz, 1H),
3.27 (d, J = 11.2 Hz, 1H), 3.29−3.24 (m, 4H), 2.99 (app t, J = 5.9 Hz,
2H), 2.17 (app td, J = 6.6, 1.8 Hz, 2H), 0.80 (s, 3H), 0.76 (s, 3H).13C
NMR (101 MHz, D2O): δ 201.1, 175.0, 173.9, 171.2, 169.0, 163.2,
162.8, 157.2, 156.4, 129.8, 129.7, 126.3, 123.5, 116.3, 116.0, 75.7, 68.4,
63.5, 60.8, 56.0, 55.5, 38.6, 38.3, 35.3, 35.1, 28.1, 20.5, 19.1. HRMS
EtOAc/Hex (24.92 g, 78%). Mp: 203 °C. [α]24 = 131.87 (c = 1.0,
D
MeOH). 1H NMR (400 MHz; DMSO-d6): δ 9.46 (br s, 1H), 7.37 (d,
J = 8.1 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 8.6 Hz, 2H),
4.98 (d, J = 8.1 Hz, 1H), 1.39 (s, 9H). 13C NMR (101 MHz, DMSO-
d6): δ 172.9, 157.1, 155.3, 129.1, 127.6, 115.2, 78.4, 57.2, 28.3. HRMS
(FAB): calcd for C13H18NO5 268.11850, found 268.11832 [M + H]+.
N-(tert-Butyloxycarbonyl)-L-(p-hydroxyphenyl)glycine-L-arginine-
(Pbf) Benzyl Ester (15). In a 250 mL round-bottomed flask equipped
with a magnetic stir bar was dissolved compound 13 was dissolved in
60 mL of freshly distilled THF. Reagent grade piperdine (6 mL) was
added, and the solution was stirred at room temperature for 30 min.
The contents of the flask were concentrated in vacuo, and excess
piperdine was removed by azeotropic distillation with toluene (2 ×
100 mL) and further dried under high vacuum to afford a white solid.
After 30 min, the solid was dissolved in 15 mL of freshly distilled
DCM and cooled to 0 °C with an ice bath.
In a separate 250 mL round-bottomed flask, 14 (1.94 g, 7.26 mmol)
was dissolved in 15 mL of freshly distilled DCM and cooled to 0 °C in
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an ice bath, and DIEA (3.16 mL, 18.15 mmol) was added. When the
solution was sufficiently cooled, PyBOP (3.78 g, 7.26 mmol) was
added, and the solution containing the free amine corresponding to
compound 13 was transferred dropwise over 3 min. The reaction was
stirred at 0 °C to room temperature over 3 h. The solution was
concentrated in vacuo, redissolved in 200 mL of EtOAc, and washed
with satd aq NH4Cl (2 × 30 mL), satd aq NaHCO3 (2 × 30 mL) and
1 × 30 mL brine. The organic solution was concentrated in vacuo, and
the product was purified by silica gel chromatography using a gradient
70:30 EtOAc/Hex to 80:20 EtOAc/Hex over 2 L. The desired product
N-(tert-Butyloxycarbonyl)-L-(p-hydroxyphenyl)glycine-L-arginine-
(Pbf)-D-[p-(benzyloxy)phenyl]glycine-L-serine-D-[p-(benzyloxy)-
phenyl]glycine Benzyl Ester (16b). The title compound was prepared
and purified analogously to compound 16a by replacing 10a with 10b
(1.17 g, 1.51 mmol). The product was obtained as a white foam (722
mg, 36%). [α]23 = 7.3 (c = 1.0, MeOH). 1H NMR (400 MHz;
D
DMSO-d6): δ 9.39 (s, 1H), 8.79 (d, J = 7.4 Hz, 1H), 8.45 (d, J = 7.6
Hz, 1H), 8.39 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 7.8 Hz, 1H), 7.45−7.18
(m, 20 H), 7.19 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.92 (d, J
= 8.8 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H), 6.40 (br s, 2H), 5.54 (d, J =
7.7 Hz, 1H), 5.46 (d, J = 7.2 Hz, 1H), 5.17−5.06 (m, 7H), 4.85 (t, J =
6.6 Hz, 1H), 4.52−4.02 (m, 2H), 3.46−3.38 (m, 2H), 3.04−2.99 (m,
2H), 2.94 (s, 2H), 2.49 (s, 3H), 2.43 (s, 3H), 2.00 (s, 3H), 1.62−1.60
(m, 1H), 1.50−1.41 (m, 3H), 1.40 (s, 6H), 1.37 (s, 9H). 13C NMR
(101 MHz, DMSO-d6): δ 170.62, 170.55, 170.5, 170.4, 169.8, 169.7,
158.2, 157.8, 157.7, 157.4, 156.8, 156.0, 137.3, 137.1, 137.0, 135.7,
131.4, 128.94, 128.91, 128.5, 128.43, 128.36, 128.2, 127.99, 127.98,
127.9, 127.8, 127.68, 127.65, 127.6, 127.5, 124.3, 116.3, 115.0, 114.9,
114.9, 114.8, 114.5, 114.4, 86.3, 69.21, 69.19, 66.2, 61.8, 42.5, 30.7,
28.3, 28.2, 19.0, 17.6, 12.3. HRMS (FAB): calcd for C72H82N8NaO15S
1353.55181, found 1353.5527 [M + Na]+.
L-(p-Hydroxyphenyl)glycine-L-arginine-D-(p-hydroxyphenyl)-
glycine-L-serine-L/D-(p-hydroxyphenyl)glycylpantetheine (17a/17b).
In a 250 mL pressure flask was dissolved protected peptide 16a or 16b
(213 mg, 0.16 mmol) in 10 mL of reagent-grade THF, to the solution
was added a catalytic amount of Pd-OH/C, and the flask was
vigorously shaken under 50 psi of H2 for 12 h. The contents of the
flask were filtered through Celite, washed with 100 mL of THF, and
concentrated in vacuo to a white foam, which was used without further
purification.
was isolated as a white foam (1.99 g, 43%). [α]25 = 31.8 (c = 1.0,
D
EtOAc). 1H NMR (400 MHz; DMSO-d6): δ 9.38 (s, 1H), 8.48 (d, J =
7.4 Hz, 1H), 7.37−7.23 (m, 5H), 7.19 (d, J = 8.6 Hz, 2H), 7.09 (d, J =
8.8 Hz, 1H), 6.69 (d, J = 8.6 Hz, 2H), 6.45 (br s, 1H), 5.14 (d, J = 8.8
Hz, 1H), 5.03−5.01 (m, 2H), 4.31 (br q, J = 6.7 Hz, 1H), 3.05 (q, J =
6.5 Hz, 2H), 2.50 (s, 3H), 2.45 (s, 3H), 2.01 (s, 3H), 1.78−1.66 (m,
1H), 1.65−1.55 (m, 1H), 1.43 (s, 3H), 1.40 (s, 3H), 1.38 (s, 9H). 13C
NMR (101 MHz, DMSO-d6): δ 171.88, 171.1, 157.9, 157.3, 156.5,
155.3, 137.7, 136.2, 131.9, 129.2, 128.9, 128.9, 128.5, 128.4, 128.2,
124.8, 116.8, 115.4, 86.7, 78.8, 66.4, 57.4, 52.3, 43.0, 31.1, 28.8, 28.6,
19.5, 18.1, 12.8. HRMS (FAB): calcd for C39H52N5O9S 766.34858,
found 766.34757 [M + H]+.
N-(tert-Butyloxycarbonyl)-L-(p-hydroxyphenyl)(glycine-L-
arginine(Pbf)-D-[p-(benzyloxy)phenyl]glycine-L-serine-L-[p-
(benzyloxy)phenyl]glycine Benzyl Ester (16a). In a 250 mL round-
bottomed flask equipped with a magnetic stir bar was dissolved
compound 15 (1.15 g, 1.31 mmol) in 30 mL of reagent-grade THF.
To this solution was added a catalytic amount of Pd-OH/C, and the
dipeptide was hydrogenated under 1 atm of H2 for 12 h. The mixture
was filtered through Celite and washed with THF (3 × 50 mL), and
the organic filtrate was concentrated in vacuo and used in the next
reaction without further purification. In a separate 250 mL round-
bottomed flask equipped with a magnetic stir bar was dissolved
tripeptide 10a (1.11 g, 1.43 mmol) in 60 mL of TFA and the mixture
stirred at room temperature for 30 min. The solution was concentrated
in vacuo, and the solvents were removed by azeotropic distillation with
toluene (3 × 50 mL).
In a 25 mL round-bottomed flask, equipped with a magnetic stir
bar, was dissolved the freshly deprotected pentapeptide in 5 mL of
reagent-grade DMF. To this solution were added DIEA (84 μL, 0.48
mmol) and PyBOP (100 mg, 0.19 mmol) followed by 11 (61 mg, 0.19
mmol). The reaction was stirred at room temperature for 1 h. The
solution was diluted with 50 mL of EtOAc, washed with satd aq
NH4Cl (2 × 15 mL) and satd aq NaHCO3 (1 × 15 mL), and
concentrated in vacuo. The residue was redissolved in 2.0 mL of 1:1
ACN/H2O solution and purified according to prep method B as a
mixture of diastereomers. The product was collected, frozen on dry ice,
and lyophilized to dryness. The lyophilized powder was dissolved in
TFA for 10 min, concentrated in vacuo, redissolved in 2 mL of 80:20
H2O/ACN supplemented with 0.1% TFA, and purified using prep
method B. Product 17a/17b product was collected, frozen on dry ice,
lyophilized to dryness, and obtained as the white TFA salt (60.8 mg,
35%). The product was further purified using Analytical Method A and
verified to be an inseparable mixture of diastereomers: 1H NMR (17a)
(400 MHz; D2O): δ 7.24 (d, J = 8.8 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H),
7.11 (d, J = 8.8 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.8 Hz,
2H), 6.76 (d, J = 8.4 Hz, 2H), 5.50 (s, 1H), 5.29 (s, 1H), 5.05 (s, 1H),
4.44 (t, J = 5.5 Hz, 1H), 4.32−4.29 (m, 1H), 3.91 (s, 1H), 3.82−3.80
(m, 2H), 3.44 (d, J = 11.2 Hz, 1H), 3.34−3.27 (m, 4H), 3.33 (d, J =
11.2 Hz), 3.06−2.99 (m, 4H), 2.26−2.22 (m, 2H), 1.70−1.64 (m,
To a third 250 mL round-bottomed flask equipped with a magnetic
stir bar was added the freshly deprotected compound 15 in 30 mL of
reagent-grade DMF. To this was added DIEA (228 μL, 1.31 mmol),
and the solution was cooled to 0 °C in an ice bath. The freshly
deprotected peptide corresponding to compound 10a was dissolved in
20 mL of reagent-grade DMF, DIEA (456 μL, 2.62 mmol) was added,
and the solution was cooled to 0 °C in an ice bath. After both solutions
were sufficiently cooled, PyBOP (747 mg, 1.43 mmol) was added to
the flask containing freshly deprotected dipeptide 15, and after 1 min,
freshly deblocked 10a was added dropwise over 2 min. The reaction
mixture was stirred from 0 °C to room temperature over 3 h. The
reaction mixture was diluted with 150 mL of EtOAc and washed with
satd aq NH4Cl (2 × 75 mL), satd aq NaHCO3 (2 × 75 mL), and brine
(1 × 75 mL). The organic solution was concentrated in vacuo, and the
product was purified by silica gel chromatography with 98:2 EtOAc/
MeOH to afford the product as a white foam (733 mg, 42%). [α]25
=
D
1
1
−20.7 (c = 1.0, MeOH). H NMR (400 MHz; DMSO-d6): δ 9.40 (s,
1H), 8.77 (d, J = 7.2 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 7.6
Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.46−7.22 (m, 20H), 7.18 (d, J =
8.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 6.69
(d, J = 8.4 Hz, 2H), 6.49−6.36 (br s, 2H), 5.51 (d, J = 8.0 Hz, 1H),
5.43 (d, J = 6.8 Hz, 1H), 5.13, 5.10 (ABq, JAB ∼ 12.1 Hz, 2H), 5.12 (s,
2H), 5.11−5.09 (m, 1H), 5.05 (s, 2H), 4.82 (t, J = 5.3 Hz, 1H), 4.43−
4.37 (m, 2H), 3.54−3.47 (m, 2H), 3.03−3.00 (m, 2H), 2.94 (s, 2H),
2.47 (s, 3H), 2.42 (s, 3H), 2.00 (s, 3H), 1.63−1.57 (m, 1H), 1.51−
1.39 (m, 3H), 1.39 (s, 6H), 1.36 (s, 9H). 13C NMR (101 MHz,
DMSO-d6): δ 170.6, 170.4, 170.4, 169.9, 169.7, 158.3, 157.7, 157.4,
156.8, 156.0, 137.3, 137.1, 137.0, 135.8, 131.4, 131.1, 129.2, 129.0,
128.4, 128.42, 128.37, 128.3, 128.1, 128.0, 128.0, 127.84, 127.81,
127.7, 127.6, 127.5, 124.3, 114.99, 114.88, 114.4, 86.3, 78.4, 69.2, 66.1,
61.69, 61.65, 55.8, 54.9, 45.89, 45.85, 42.5, 28.3, 28.2, 26.0, 25.9, 19.0,
17.6, 12.3. HRMS (FAB): calcd for C72H83N8O15S 1331.56986, found
1331.57123 [M + H]+.
2H), 1.49−1.40 (m, 2H), 0.85 (s, 3H), 0.81 (s, 3H). H NMR (17b)
(400 MHz; D2O): δ 7.30 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H),
7.16 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.7 Hz,
2H), 6.76 (d, J = 8.6 Hz, 2H), 5.45 (s, 1H), 5.28 (s, 1H), 5.03 (s, 1H),
4.44 (t, J = 5.5 Hz, 1H), 4.32−4.29 (m, 1H), 3.91 (s, 1H), 3.79−3.78
(m, 2H), 3.44 (d, J = 11.2 Hz, 1H), 3.34−3.27 (m, 4H), 3.33 (d, J =
11.2 Hz), 3.06−2.99 (m, 4H), 2.24 (J = 6.00, 2H), 1.70−1.64 (m, 2H),
1.49−1.40 (m, 2H), 0.85 (s, 3H), 0.81 (s, 3H). 13C NMR (17a/17b)
(101 MHz, D2O): δ 201.0, 175.0, 173.9, 172.6, 172.1, 171.4, 168.9,
168.8, 163.5, 163.1, 162.8, 162.4, 157.3, 156.6, 156.4, 156.3, 156.1,
129.7, 129.7, 129.6, 129.6, 129.3, 129.2, 129.2, 127.1, 126.4, 123.2,
123.2, 117.8, 116.3, 115.98, 115.95, 114.9, 75.8, 68.4, 63.6, 63.5, 63.4,
60.8, 57.5, 57.2, 55.9, 55.6, 54.0, 40.5, 40.4, 38.6, 38.4, 35.3, 35.2, 28.1,
27.7, 24.3, 20.5, 19.1. HRMS (FAB): calcd for C44H61N10O13S
969.41403, found 969.41241 [M + H]+.
N-(tert-Butyloxycarbonyl)-D-[p-(benzyloxy)phenyl]glycine-L-tert-
butylphosphoserine-L-benzyl-[p-(benzyloxy)phenyl]glycine Benzyl
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Ester (18a). In a 100 mL flame-dried round-bottomed flask, equipped
with a magnetic stir bar and an argon inlet, was dissolved protected
tripeptide 10a (2.10 g, 2.71 mmol) in 15 mL of freshly distilled THF.
To this solution was added tetrazole (7.22 mL, 0.45 M in ACN, 3.25
mmol) followed by di-tert-butyl N,N-diisopropylphosphoramidite
(1.03 mL, 3.25 mmol). The reaction mixture was allowed to stir at
room temperature for 12 h. The solution was cooled to 0 °C in an ice
bath, tert-butyl hydroperoxide (590 μL, 5.5 M in decane, 3.25 mmol)
was added, and the reaction was stirred at 0 °C for 3 h. The solution
was diluted with 150 mL of EtOAc and washed with 1:1 satd aq
NaHCO3/NaS2O3 (2 × 50 mL). The EtOAc was concentrated in
vacuo to a viscous yellow oil, which was purified by flash
chromatography on silica gel that was pretreated for 12 h with a 1:1
solution of EtOAc/Hex containing 5% triethylamine. The product was
eluted with a step-gradient mobile phase of 70:30 Hex/EtOAc to
50:50 Hex/EtOAc to elute the partially purified product as a colorless
oil containing phosphoramidite impurities. Crystallization of the oil in
collected, frozen on dry ice, and lyophilized to dryness as a white TFA
salt (23.0 mg, 25%). The product was further purified using Analytical
Method A and verified to be an inseparable mixture of diastereomers.
1H NMR (19a) (400 MHz; D2O): δ 7.31 (d, J = 8.7 Hz, 2H), 7.23 (d,
J = 8.6 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H),
5.45 (s, 1H), 5.08 (s, 1H), 4.57 (t, J = 5.4 Hz, 1H), 3.97−3.89 (m,
2H), 3.41 (d, J = 11.3 Hz, 1H), 3.30−3.25 (m, 5H), 3.05−2.98 (m,
2H), 2.18 (app td, J = 6.6, 2.6 Hz, 2H), 0.81 (s, 3H), 0.77 (s, 3H). 1H
NMR (19b) (400 MHz; D2O): δ 7.31 (d, J = 8.7 Hz, 2H), 7.25 (d, J =
8.5 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.82 (d, J = 8.9 Hz, 2H), 5.46 (s,
1H), 5.06 (s, 1H), 4.51 (t, J = 5.2 Hz, 1H), 4.05 (t, J = 6.1 Hz, 1H),
3.97−3.89 (m, 1H), 3.40 (d, J = 11.3 Hz, 1H), 3.30−3.25 (m, 5H),
3.05−2.98 (m, 2H), 2.18 (app td, J = 6.6, 2.6 Hz, 2H), 0.81 (s, 3H),
0.77 (s, 3H). 13C NMR (19a/19b) (101 MHz, D2O): δ 201.4, 200.9,
175.0, 173.9, 170.6, 168.9, 163.1, 162.8, 157.2, 156.4, 130.0, 129.8,
129.7, 126.23, 126.18, 123.4, 123.3, 116.4, 116.0, 75.7, 68.4, 63.9, 63.7,
56.1, 56.0, 54.2, 38.6, 38.4, 35.3, 35.1, 28.1, 20.5, 19.1. 31P NMR (19a)
(162 MHz; D2O): δ −0.04. 31P NMR (19b) (162 MHz; D2O): δ
−0.074. HRMS (FAB): calcd for C30H43N5O13 PS 744.23157, found
744.23037 [M + H]+.
Et2O and Hex provided the desired product as a white granular solid
1
(1.67 g, 64%). Mp: 84 °C. [α]24 = −3.2 (c = 1.0, EtOAc). H NMR
D
(400 MHz; CDCl3): δ 7.76 (br s, 1H), 7.68 (br s, 1H), 7.43−7.21 (m,
20H), 6.94 (d, J = 8.8, 2H), 6.93 (d, J = 8.8, 2H), 5.63 (d, J = 6.0 Hz,
1H), 5.57 (d, J = 6.8 Hz, 1H), 5.16 (s, 1H), 5.15 (s, 1H), 5.06 (s, 2H),
5.05 (s, 2H), 4.69−4.66 (m, 1H), 4.42 (br t, J = 8.8 Hz, 1H), 3.96 (td,
J = 10.1, 3.7 Hz, 1H), 1.41 (s, 9H), 1.40 (s, 9H), 1.37 (s, 9H). 13C
NMR (101 MHz, CDCl3): δ 171.3, 171.3, 170.2, 168.3, 159.0, 136.9,
135.5, 129.0, 128.7, 128.60, 128.56, 128.3, 128.2, 128.0, 128.0, 127.6,
127.6, 115.4, 115.2, 100.1, 83.8, 83.7, 83.5, 83.4, 70.1, 67.3, 65.8, 56.4,
53.7, 53.6. 31P NMR (162 MHz, CDCl3): δ −8.92. HRMS (FAB):
calcd for C53H65N3O12P 966.43059, found 966.42975 [M + H]+.
N-(tert-Butyloxycarbonyl)-D-[p-(benzyloxy)phenyl]glycine-L-tert-
butylphosphoserine-D-[p-(benzyloxy)phenyl]glycine Benzyl Ester
(18b). The title compound was prepared and purified analogously to
compound 18a by replacing 10a with 10b (1.41 g, 1.82 mmol). The
product was obtained as a white foam (1.09 g, 62%). Mp: 83 °C.
[α]24D = −49.7 (c = 1.0, EtOAc). 1H NMR (400 MHz; CDCl3): δ 7.81
(br s, 2H), 7.44−7.22 (m, 20H), 6.94 (d, J = 8.4 Hz, 2H), 6.92 (d, J =
8.4 Hz, 2H), 5.55 (d, J = 7.1 Hz, 1H), 5.53 (br s, 1H), 5.19, 5.14 (ABq,
JAB = 12.4 Hz, 2H) 5.05 (2s, 4H), 4.70−4.66 (m, 1H), 4.43 (br t, J =
10.2 Hz, 1H), 3.92 (br dt, J = 9.2, 4.5 Hz, 1H), 1.44 (s, 9H), 1.37 (s,
18H). 13C NMR (101 MHz, CDCl3): δ 171.32, 171.31, 170.26, 168.3,
159.02, 158.96, 137.0, 136.9, 135.5, 128.8, 128.7, 128.6, 128.4, 128.2,
128.14, 128.12, 128.07, 127.6, 115.4, 115.2, 83.9, 83.8, 83.5, 70.13,
70.11, 67.3, 65.8, 56.5, 53.6, 53.6, 29.9, 29.8, 29.8, 28.4. 31P NMR (162
MHz, CDCl3): δ −8.85. HRMS (FAB): calcd for C53H65N3O12P
966.43059, found 966.43025 [M + H]+.
D-(p-Hydroxyphenyl)glycine-L-phosphoserine-L-(p-hydroxy-
phenyl)glycylpantetheine (19a/19b). In a 250 mL pressure flask was
dissolved protected peptide 18a or 18b (77 mg, 0.10 mmol) in 10 mL
of reagent-grade THF, to it was added a catalytic amount of Pd-OH/
C, and the flask was vigorously shaken under 50 psi of H2 for 12 h.
The mixture was filtered through Celite, washed with 100 mL of THF,
concentrated in vacuo to a white foam, and used without further
purification.
In a 10 mL round-bottomed flask equipped with a magnetic stir bar
was dissolved freshly hydrogenolyzed tripeptide corresponding to
compounds 18a or 18b (50.35 mg, 0.10 mmol) in 2 mL of reagent-
grade DMF, and to this solution was added K2CO3 (42 mg, 0.30
mmol) and th mixture cooled to 0 °C in an ice bath. To this cooled
solution were added PyBOP (63 mg, 0.12 mmol) and 11 (39 mg, 0.12
mmol), and the reaction mixture was stirred for 30 min at room
temperature. The solution was diluted with 50 mL of EtOAc and
washed with satd aq NH4Cl (2 × 20 mL), satd aq NaHCO3 (1 × 20
mL), and brine (1 × 20 mL). The EtOAc extract was concentrated in
vacuo to a viscous oil, which was redissolved in 2 mL 70:30 ACN/
H2O. The product was purified by prep method B, collected on dry
ice, and lyophilized to dryness. The freshly lyophilized product was
dissolved in 5 mL of reagent-grade TFA and stirred at room
temperature for 15 min. The TFA was removed in vacuo, and the
residue was resuspended in 2 mL of 70:30 H2O/ACN supplemented
with 0.1% TFA. The reaction mixture was purified by prep method A,
N-(tert-Butyloxycarbonyl)-L-acetylserine-L-[p-(benzyloxy)phenyl]-
glycine Benzyl Ester (20a). In a flame-dried 250 mL round-bottomed
flask equipped with a magnetic stir bar and an argon inlet was
dissolved protected tripeptide 10a (4.00 g, 5.17 mmol) in 40 mL of
reagent-grade pyridine, to this was added acetic anhydride (537 μL,
5.69 mmol), and the reaction was stirred at room temperature for 12 h.
The reaction mixture was concentrated in vacuo to a yellow solid,
which was purified by silica gel flash chromatography utilizing an
isocratic mobile phase 70: 30 Hex/EtOAc to afford the product as a
1
white solid (4.18 g, 99%). [α]23 = −4.4 (c = 1.0, EtOAc). H NMR
D
(400 MHz; DMSO-d6): δ 8.88 (d, J = 6.3 Hz, 1H), 8.48 (d, J = 8.0 Hz,
1H), 7.45−7.21 (m, 20H), 7.02 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6
Hz, 2H), 5.41 (d, J = 6.4 Hz, 1H), 5.21 (d, J = 7.7 Hz, 1H), 5.16−5.08
(2s + ABq, 6H), 4.70 (br q, J = 7.0 Hz, 1H), 4.09−4.04 (m, 2H), 1.78
(s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ 170.6,
170.2, 169.9, 168.5, 158.4, 157.7, 154.8, 137.1, 137.0, 135.7, 130.9,
129.2, 128.5, 128.4, 128.4, 128.4, 128.0, 127.9, 127.8, 127.62, 127.59,
127.5, 114.9, 114.4, 78.4, 69.2, 69.1, 66.2, 63.2, 56.9, 56.0, 51.1, 28.2,
20.3. HRMS (FAB): calcd for C47H50N3O10 816.34620, found
816.34819 [M + H]+.
N-(tert-Butyloxycarbonyl)-L-acetylserine-D-benzyl-[p-(benzyloxy)-
phenyl]glycine (20b). The title compound was prepared and purified
analogously to compound 20a by replacing 10a with 10b (4.00 g, 5.17
mmol). The product was obtained as a white solid (4.18 g, 99%):
[α]23D = −51.3 (c = 1.0, EtOAc). 1H NMR (400 MHz; DMSO-d6): δ
8.93 (d, J = 6.6 Hz, 1H), 8.47 (d, J = 8.3 Hz, 1H), 7.46−7.23 (m,
20H), 7.01 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 5.44 (d, J =
7.1 Hz, 1H), 5.24 (d, J = 7.9 Hz, 1H), 5.16, 5.13 (ABq, JAB = 12.7 Hz,
2H), 5.11 (s, 2H), 5.08 (s, 2H), 4.72 (br q, J = 6.3 Hz, 1H), 4.07−3.96
(sym m, 2H), 1.73 (s, 3H), 1.36 (s, 9H). 13C NMR (101 MHz,
DMSO-d6): δ 171.1, 170.7, 170.3, 168.9, 158.8, 158.2, 137.6, 137.4,
136.2, 131.3, 129.4, 128.91, 128.89, 128.8, 128.5, 128.3, 128.3, 128.1,
128.1, 128.0, 115.4, 114.9, 69.7, 69.6, 66.7, 56.3, 51.7, 28.6, 20.7.
HRMS (FAB): calcd for C47H50N3O10 816.34620, found 816.34773
[M + H]+.
D-(p-Hydroxyphenyl)glycine-L-acetylserine-L-(p-hydroxyphenyl)-
glycylpantetheine (21a). In a 250 mL pressure flask was dissolved
protected peptide 20a (147 mg, 0.18 mmol) in 10 mL of reagent-grade
THF, and to it was added a catalytic amount of Pd-OH/C and the
mixture was vigorously shaken under 50 psi of H2 for 12 h. The
contents of the flask were filtered through Celite, washed with 100 mL
of THF, and concentrated in vacuo to a white foam and used without
further purification.
In a 10 mL round-bottomed flask equipped with a magnetic stir bar
was dissolved 20a (100 mg, 0.18 mmol) in 2 mL of reagent-grade
DMF, to this solution was added K2CO3 (76 mg, 0.55 mmol), and the
mxiture was cooled to 0 °C in an ice bath. To this cooled solution was
added PyBOP (114 mg, 0.20 mmol) followed by 11 (70 mg, 0.20
mmol), and the reaction was then stirred for 30 min at room
temperature. The solution was diluted with 50 mL of EtOAc and
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washed with satd aq NH4Cl (2 × 20 mL), satd aq NaHCO3 (1 × 20
mL), and brine (1 × 20 mL). The EtOAc was concentrated in vacuo
to a viscous oil, which was redissolved in 2 mL of 70:30 ACN/H2O.
The reaction was purified by prep method A, collected, frozen on dry
ice, and lyophilized to dryness. The product was dissolved in 5 mL of
reagent-grade TFA and stirred at room temperature for 15 min. The
TFA was removed in vacuo, and the residue was resuspended in 2 mL
of 70:30 H2O/ACN supplemented with 0.1% TFA. The freshly
deprotected product purified by prep method A was collected, frozen
on dry ice, and lyophilized to dryness to afford the product as a white
TFA salt (63.0 mg, 38%). Isolation of 2 mg of diasteromerically pure
material was achieved using analytical method A. 1H NMR (400 MHz;
D2O): δ 7.25 (2d, J = 8.2 Hz, 4H), 6.86 (2d, J = 8.2 Hz, 4H), 5.47 (s,
1H), 5.04 (s, 1H), 4.26 (d, J = 5.1 Hz, 2H), 3.87 (s, 1H), 3.40 (d, J =
11.3 Hz, 1H), 3.29−3.24 (m, 5H), 3.00 (br t, J ∼ 6.1 Hz, 2H), 2.19 (br
t, J ∼ 6.4 Hz, 2H), 1.82 (s, 3H), 0.80 (s, 3H), 0.77 (s, 3H). 13C NMR
(101 MHz, D2O): δ 200.9, 188.6, 175.0, 173.8, 173.3, 169.9, 168.9,
166.9, 157.2, 156.4, 155.4, 129.7, 126.1, 123.5, 117.8, 116.3, 116.0,
83.9, 75.7, 68.4, 63.6, 63.0, 55.9, 52.4, 38.6, 38.3, 35.3, 35.1, 28.1, 20.5,
19.9, 19.1. HRMS (FAB): calcd for C32H44N5O11S 706.27580, found
706.27413 [M + H]+.
D-(p-Hydroxyphenyl)glycine-L-acetylserine-D-(p-hydroxyphenyl)-
glycylpantetheine (21b). The title compound was prepared and
purified analogously to compound 21a by replacing 20a with 20b
(146.9 mg, 0.18 mmol). The product was obtained as a white TFA salt
(64.7 mg, 39%). Isolation of 2 mg of diasteromerically pure material
was achieved using analytical method A. 1H NMR (400 MHz; D2O): δ
7.28 (d, J = 8.7 Hz, 2H), 7.22 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.8 Hz,
2H), 6.84 (d, J = 8.8 Hz, 2H), 5.47 (s, 1H), 5.06 (s, 1H), 4.75 (X of
ABX, J = 6.4, 4.8 Hz, 1H), 4.14 (AB of ABX, J = 11.6, 6.4, 4.8 Hz, 2H),
3.87 (s, 1H), 3.40 (d, J = 11.3 Hz, 1H), 3.31−3.26 (m, 4H), 3.29 (d, J
= 11.3 Hz, 1H), 3.08−2.97 (m, 2H), 2.20 (td, J = 6.6, 2.1 Hz, 2H),
1.69 (s, 3H), 0.81 (s, 3H), 0.77 (s, 3H). 13C NMR (101 MHz, D2O): δ
201.0, 175.0, 173.9, 173.2, 169.9, 169.0, 157.3, 156.4, 129.8, 129.7,
126.5, 123.5, 116.3, 116.0, 75.7, 68.4, 63.6, 62.9, 56.0, 52.3, 38.6, 38.3,
35.3, 35.1, 20.4, 19.7, 19.1. HRMS (FAB): calcd for C32H44N5O11S
706.27580, found 706.27532 [M + H]+.
L-[p-(Benzyloxy)phenyl]glycine tert-Butyl Ester Toluenesulfonate
(22a). In a 500 mL pressure bottle equipped with a magnetic stir bar
was suspended L-[p-(benzyloxy)phenyl]glycine14 (10.00 g, 49.4 mmol)
in 100 mL of reagent-grade dioxane containing 10 mL of concentrated
sulfuric acid and the mixture cooled to 0 °C in an ice bath. In a
separate 125 mL Erlenmeyer flask, isobutylene (100 mL of liquid) was
condensed from gas at −78 °C, and the liquid was quickly added to
the pressure bottle and sealed. The contents of the pressure bottle
were stirred at room temperature for 12 h. The solution was cooled to
0 °C with an ice bath, and the solution was quickly poured into an ice-
cold mixture of 400 mL of 1.0 M NaOH and 500 mL of Et2O. The
mixture was transferred to a 1 L separatory flask, the organics were
partitioned and set aside, and the aqueous layer was washed with Et2O
(2 × 100 mL). The organic fractions were combined, dried with brine
(2 × 100 mL), and concentrated in vacuo to approximately 150 mL.
This ethereal solution was added to a separate flask containing p-
toluenesulfonic acid monohydrate (9.40 g, 49.4 mmol) in 200 mL of
Et2O, which precipitated the product. The precipitate was filtered and
J = 8.0 Hz, 2H), 7.45−7.34 (m, 7H), 7.13 (d, J = 7.0 Hz, 2H), 7.11 (d,
J = 8.7 Hz, 2H), 5.15 (s, 2H), 5.07 (br q., J = 5.4 Hz, 1H), 2.30 (s,
3H), 1.38 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ 168.2, 159.5,
146.0, 138.2, 137.2, 130.0, 128.9, 128.6, 128.4, 128.2, 128.1, 126.0,
125.5, 115.7, 100.0, 83.6, 69.7, 55.8, 27.9, 21.3. HRMS (FAB): calcd
for C19H24NO3 314.17562, found 314.17550 [M + H]+.
N-(4,6-Diphenyl-4-oxazolin-2-onyl)-L-seryl-L-[p-(benzyloxy)-
phenyl)glycine tert-Butyl Ester (23a). In a flame-dried 250 mL round-
bottomed flask equipped with a magnetic stir bar were dissolved L-Ox-
serine dicyclohexylammonium salt26 (7.50 g, 14.80 mmol) and DIEA
(2.57 mL, 14.80 mmol) in 25 mL of reagent-grade DMF and cooled to
0 °C in an ice bath. In a separate round-bottomed flask, 22a (8.62 g,
17.76 mmol) was dissolved in 10 mL of reagent-grade DMF, to it was
added DIEA (2.57 mL, 14.80 mmol), and the mxiture was cooled to 0
°C. Once both solutions were sufficiently cooled, PyBOP (9.24 g,
17.76 mmol) was added to the flask containing L-Ox-serine, and after 1
min the flask containing 22a was transferred dropwise over 2 min to
the activated acid. The reaction was stirred for 3 h from 0 °C to room
temperature. The solution was diluted with 200 mL of EtOAc and
washed with satd aq NH4Cl (2 × 75 mL), satd aq NaHCO3 (2 × 75
mL), and brine (1 × 75 mL). The EtOAc was concentrated in vacuo
to a viscous yellow oil, and the product was purified as a white foam by
silica gel chromatography with 60:40 Hex/EtOAc to afford the
product as a white solid (6.52 g, 71%). [α]25D = 39.8 (c = 1.0, EtOAc).
1H NMR (400 MHz; DMSO-d6): δ 8.73 (d, J = 6.8 Hz, 1H), 7.57−
7.22 (m, 16H), 7.12 (d, J = 7.0 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 5.28
(br s, 1H), 5.20 (d, J = 6.8 Hz, 1H), 5.12 (s, 2H), 4.21 (dd, J = 9.0, 5.8
Hz, 1H), 4.06−3.99 (m, 1H), 3.91 (dt, J = 11.2, 5.7 Hz, 1H), 1.35 (s,
9H).13C NMR (101 MHz, DMSO-d6): δ 169.7, 166.7, 158.6, 153.8,
137.5, 133.5, 131.3, 130.8, 129.9, 129.4, 129.2, 128.9, 128.9, 128.3,
128.14, 128.08, 127.1, 125.2, 124.1, 115.3, 81.7, 69.7, 59.4, 58.5, 57.1,
28.0. HRMS (FAB): calcd for C37H36N2O7 620.25225, found
620.25150 [M]+.
N-(4,6-Diphenyl-4-oxazolin-2-onyl)-L-seryl-D-[p-(benzyloxy)-
phenyl)glycine tert-Butyl Ester (23b). The title compound was
prepared and purified analogously to compound 23a by replacing 22a
with 22b (8.62 g, 17.76 mmol). The product was obtained as a white
foam (6.34 g, 69%). [α]23D = −50.5 (c = 1.0, EtOAc). 1H NMR (400
MHz; DMSO-d6): δ 8.68 (d, J = 7.2 Hz, 1H), 7.59−7.23 (m, 15H),
7.15 (d, J = 7.0 Hz, 2H), 7.03 (d, J = 8.8 Hz, 2H), 5.43 (t, J = 5.6 Hz,
1H), 5.22 (d, J = 7.2 Hz, 1H), 5.12 (s, 2H), 4.23 (dd, J = 8.3, 6.3 Hz,
1H), 4.05−3.99 (m, 1H), 3.86 (dt, J = 11.3, 5.8 Hz, 1H), 1.34 (s,
9H).13C NMR (101 MHz, DMSO-d6): δ 169.8, 167.1, 158.6, 153.8,
137.5, 133.5, 131.3, 130.8, 130.0, 129.2, 129.2, 128.9, 128.9, 128.3,
128.2, 128.1, 128.1, 127.0, 125.2, 124.2, 115.3, 81.7, 69.7, 59.3, 58.6,
57.1, 28.0. HRMS (FAB): calcd for C37H36N2O7 620.25225, found
620.25214 [M]+.
3-N-(4,5-Diphenyl-4-oxazolin-2-onyl)-epi-aminonocardicin tert-
Butyl Ester (24a). In a flame-dried 100 mL round-bottomed flask
equipped with a magnetic stir bar was dissolved 23a (3.28 g, 5.28
mmol) in 30 mL of freshly distilled THF, and the flask was wrapped
with aluminum foil to exclude light. To this solution were added
P(OEt)3 (1.09 mL, 6.34 mmol) and DEAD (994 μL, 6.34 mmol), and
the reaction was stirred at room temperature for 12 h. The THF was
removed in vacuo, and the residue was purified by flash
chromatography with an isocratic mobile phase of 60:40 EtOAc/
dried under high vacuum to afford the product as a white salt (15.35 g,
64%). [α]25 = 44.4 (c = 1.0, MeOH); H NMR (400 MHz; DMSO-
Hex to afford the product as a white foam (2.99 g, 94%). [α]22
=
1
D
D
1
d6 δ 8.68 (br s, 3H), 7.50 (d, J = 8.0 Hz, 2H), 7.45−7.34 (m, 7H), 7.13
(d, J = 7.0 Hz, 2H), 7.11 (d, J = 8.7 Hz, 2H), 5.15 (s, 2H), 5.07 (br q.,
J = 5.4 Hz, 1H), 2.30 (s, 3H), 1.38 (s, 9H). 13C NMR (101 MHz,
DMSO-d6): δ 168.2, 159.5, 146.0, 138.2, 137.2, 130.0, 128.9, 128.6,
128.4, 128.2, 128.1, 126.0, 125.5, 115.7, 100.0, 83.6, 69.7, 55.8, 27.9,
21.3. HRMS (FAB): calcd for C19H24NO3 314.17562, found
314.17537 [M + H]+.
−99.6 (c = 1.0, EtOAc). H NMR (400 MHz; DMSO-d6): δ 7.58−
7.17 (m, 17H), 7.02 (d, J = 8.7 Hz, 2H), 5.27 (s, 1H), 5.09 (s, 2H),
4.82 (dd, J = 5.5, 2.7 Hz, 1H), 3.97 (dd, J = 5.6, 2.7 Hz, 1H), 3.50 (t, J
= 5.7 Hz, 1H), 1.45 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ
167.2, 163.1, 158.3, 152.0, 136.9, 133.6, 130.7, 130.4, 129.6, 128.8,
128.7, 128.4, 128.0, 127.8, 127.7, 127.2, 126.2, 125.5, 124.0, 123.4,
114.9, 82.2, 69.2, 57.7, 56.6, 45.1, 27.5. HRMS (FAB): calcd for
C37H34N2O6 602.24169, found 602.24039 [M]+.
3-N-(4,5-Diphenyl-4-oxazolin-2-onyl)aminonocardicin tert-Butyl
Ester (24b). The title compound was prepared and purified
analogously to compound 24a by replacing 23a with 23b (3.28 g,
5.28 mmol). The product was obtained as a white solid (3.05 g, 96%).
[α]23D = −116.3 (c = 1.0, EtOAc). 1H NMR (400 MHz; DMSO-d6): δ
D-[p-(Benzyloxy)phenyl]glycine tert-Butyl Ester Toluenesulfonate
(22b). The title compound was prepared and purified analogously to
compound 22a by replacing L-[p-(benzyloxy)phenyl]glycine with D-[p-
(benzyloxy)phenyl]glycine14 (10.0 g, 49.4 mmol). The product was
obtained as a white salt (15.59 g, 65%). [α]25 = −45.7 (c = 1.0,
D
MeOH). 1H NMR (400 MHz; DMSO-d6): δ 8.68 (br s, 3H), 7.50 (d,
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7.59−7.14 (m, 17H), 7.05 (d, J = 8.8 Hz, 2H), 5.26 (s, 1H), 5.14 (s,
2H), 4.84 (dd, J = 5.7, 2.8 Hz, 1H), 3.69 (t, J = 5.7 Hz, 1H), 3.40 (dd,
J = 5.7, 2.8 Hz, 1H), 1.38 (s, 9H).13C NMR (101 MHz, DMSO-d6): δ
167.9, 163.3, 158.4, 152.1, 136.9, 133.7, 130.7, 130.6, 129.5, 129.3,
128.7, 128.4, 128.0, 127.9, 127.8, 127.1, 125.4, 125.2, 124.0, 123.2,
115.0, 82.2, 69.3, 57.6, 56.1, 44.8, 27.5. HRMS (FAB): calcd for
C37H34N2O6 602.24169, found 602.24038 [M]+.
the mixed anhydride. The reaction mixture was stirred at 0 °C for 30
min, warmed to room temperature by removal of the ice bath, and
stirred for an additional 3 h. The solution was diluted with 150 mL of
EtOAc and washed with satd aq NH4Cl (2 × 40 mL), satd aq
NaHCO3 (1 × 40 mL) ,and brine (1 × 40 mL). The dried EtOAc
extract was concentrated in vacuo to a viscous yellow oil, which was
purified by silica gel flash chromatography using an isocratic mobile
phase 60:40 EtOAc/Hex to afford the product as a white foam (832
epi-3-Aminonocardicin tert-Butyl Ester Hydrochloride (25a). In a
250 mL pressure bottle was dissolved 24a (3.00 g, 4.98 mmol) in 15
mL of reagent-grade THF, and to this was added a catalytic amount of
Pd-OH/C. Hydrogenolysis was carried out over 48 h under 50 psi of
H2 on a Parr apparatus with vigorous shaking. The mixture was filtered
through Celite, and the Pd-OH/C was washed with THF (3 × 50
mL). The filtrate was concentrated in vacuo to a viscous oil, which was
partitioned into 30 mL of Et2O and 10 mL of water-containing concd
HCl (457 μL, 5.48 mmol). The aqueous layer was washed with Et2O
(2 × 30 mL), flash frozen, and lyophilized to dryness to afford the
1
mg, 64%). [α]22 = 0.80 (c = 1.0, MeOH). H NMR (400 MHz;
D
DMSO-d6): δ 9.60 (s, 1H), 9.39 (s, 1H), 8.87 (d, J = 7.9 Hz, 1H), 7.17
(d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 6.76 (d, J = 8.6 Hz, 2H),
6.68 (d, J = 8.6 Hz, 2H), 5.25 (s, 1H), 5.08 (d, J = 8.6 Hz, 1H), 4.81
(ddd, J = 7.8, 5.2, 2.7 Hz, 1H), 3.30 (t, J = 5.3 Hz, 1H), 3.26 (dd, J =
4.1, 2.2), 1.39 (s, 9H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO-d6):
δ 171.3, 168.6, 166.3, 157.9, 157.3, 155.3, 129.5, 129.1, 128.9, 124.6,
116.1, 115.4, 82.4, 78.8, 57.9, 55.4, 28.7, 28.0. HRMS (FAB): calcd for
C28H36N3O8 542.25024, found 542.2496 [M + H]+.
product as the white HCl salt (1.46 g, 89%). [α]24 = 101.9 (c = 1.0,
Protected Nocardicin G (27b). The title compound was prepared
and purified analogously to compound 27a by replacing 25a with 25b
D
1
MeOH). H NMR (400 MHz; DMSO-d6): δ 9.76 (s, 1H), 9.04 (s,
(789 mg, 2.40 mmol). The product was obtained as a white foam (871
3H), 7.14 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 5.30 (s, 1H),
4.48 (dd, J = 5.4, 2.4 Hz, 1H), 3.58 (dd, J = 6.3, 2.4 Hz, 1H), 3.43 (t, J
= 6.2 Hz, 1H), 1.42 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ
167.7, 162.1, 157.8, 129.0, 123.8, 115.7, 82.4, 57.8, 53.3, 44.2, 27.6.
HRMS (FAB): calcd for C15H21N2O4 293.15013, found 293.14937 [M
+ H]+.
1
mg, 67%). [α]24 = −141.0 (c = 1.0, MeOH). H NMR (400 MHz;
D
DMSO-d6): δ 9.63 (s, 1H), 9.39 (s, 1H), 8.77 (d, J = 8.1 Hz, 1H), 7.13
(d, J = 8.4 Hz, 4H), 6.78 (d, J = 8.6 Hz, 3H), 6.67 (d, J = 8.6 Hz, 3H),
5.29 (s, 1H), 5.00 (d, J = 8.4 Hz, 1H), 4.88−4.85 (m, 1H), 3.67 (t, J =
5.3 Hz, 1H), 2.88−2.87 (m, 1H), 1.40 (s, 9H), 1.36 (s, 9H). 13C NMR
(101 MHz, DMSO-d6): δ 171.2, 168.8, 166.6, 158.0, 157.3, 155.3,
129.8, 129.1, 128.9, 124.3, 116.1, 115.4, 82.4, 78.8, 57.9, 55.1, 28.6,
28.0. HRMS (FAB): calcd for C28H36N3O8 542.25024, found
542.24950 [M + H]+.
N-(tert-Butyloxycarbonyl)-epi-nocardicin G (28a). In a 100 mL
round-bottomed flask, equipped with a magnetic stir bar, was dissolved
27a (470 mg, 0.88 mmol) in 20 mL of reagent-grade TFA and the
mixture stirred at room temperature for 30 min. The TFA was
evaporated under reduced pressure, and residual TFA was removed by
azeotropic distillation with toluene (2 × 50 mL) to provide an orange
oil. To this oil were added 10 mL of reagent grade THF, 2 mL of
water, and DIEA (459 μL, 2.63 mmol).
Aminonocardicin tert-Butyl Ester Hydrochloride (25b). The title
compound was prepared and purified analogously to compound 25a
by replacing 24a with 24b (3.00 g, 4.98 mmol). The product was
obtained as a white HCl salt (1.51 g, 92%). [α]25D = −146.2 (c = 1.0,
MeOH. 1H NMR (400 MHz; DMSO-d6): δ 9.76 (br s, 1H), 8.93 (br
s, 2H), 7.16 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 5.33 (s, 1H),
4.56 (d, J = 3.9 Hz, 1H), 3.70 (t, J = 5.8 Hz, 1H), 3.06 (dd, J = 6.1, 2.1
Hz, 1H), 1.40 (s, 9H). 13C NMR (101 MHz, DMSO-d6): δ 168.1,
162.2, 157.9, 129.5, 123.0, 115.7, 82.3, 57.7, 53.0, 43.8, 27.5. HRMS
(FAB): calcd for C15H21N2O4 293.15013, found 293.14955 [M + H]+.
N-(tert-Butyloxycarbonyl)-D-(p-hydroxyphenyl)glycine (26). In a
500 mL Erlenmeyer flask equipped with a magnetic stir bar was
dissolved D-(p-hydroxyphenyl)glycine (20.00 g, 119.57 mmol) in 300
mL of 1.0 M NaOH, and to this was added di-tert-butyl dicarbonate
(31.31 g, 143.48 mmol) in 500 mL of reagent grade THF. The
reaction was stirred at room temperature for 12 h. The THF was
partitioned with 200 mL of Et2O and separated. The aqueous layer was
cooled to 0 °C in an ice bath and acidified to pH 2.0 with concd HCl.
The acidified aqueous mixture was extracted with EtOAc (3 × 100
mL), and the organic extractions were pooled, washed with brine (1 ×
75 mL), and concentrated in vacuo to a viscous oil. The product was
crystallized from EtOAc/Hex as a white solid (29.46 g, 97%). Mp: 198
°C. [α]25D = −133.7 (c = 1.0, MeOH). 1H NMR (400 MHz; DMSO-
d6): δ 7.37 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 6.73 (d, J =
8.6 Hz, 2H), 4.98 (d, J = 8.1 Hz, 1H), 1.39 (s, 9H). 13C NMR (101
MHz, DMSO-d6): δ 172.9, 157.2, 155.3, 129.1, 127.7, 115.2, 78.4,
57.2, 28.3. HRMS (FAB): calcd for C13H18NO5 268.11850, found
268.11831 [M + H]+.
Protected epi-Nocardicin G (27a). In a flame-dried 100 mL round-
bottomed flask equipped with a magnetic stir bar and an argon inlet
was dissolved compound 26 (963 mg, 3.60 mmol) in 10 mL of freshly
distilled acetone. To this solution were added 2,6-lutidine (370 μL,
3.26 mmol) and a catalytic amount of N-methylmorpholine (79 μL,
0.72 mmol). The solution was cooled to −50 °C with the aide of a dry
ice/acetone bath. Once the solution was sufficiently cold, isobutyl
chloroformate (427 μL, 3.26 mmol), dissolved in 2 mL of freshly
distilled acetone, was added dropwise to the solution containing 26
over 2 min. The solution was stirred at −50 to −30 °C for 30 min
during which time a precipitate formed. The dry ice/acetone bath was
removed and replaced with an ice bath, and the mixture was stirred at
0 °C for an additional 20 min.
In a separate 50 mL round-bottomed flask was dissolved di-tert-
butyl dicarbonate (227 mg, 1.04 mmol) in 5 mL of THF and the
solution transferred dropwise over 2 min to the flask containing freshly
deprotected 27a. The reaction was stirred at room temperature for 12
h. The solution was diluted with 50 mL of EtOAc and washed with
satd aq NH4Cl (2 × 20 mL). The EtOAc was concentrated in vacuo to
yield a yellow foam. The yellow foam was purified by flash silica gel
chromatography, eluting with an isocratic mobile phase 70:30 EtOAc/
Hex supplemented with 1% acetic acid. The product was obtained as a
light yellow solid (421 mg, 73%). [α]22D = 17.7 (c = 1.0, MeOH). 1H
NMR (400 MHz; DMSO-d6): δ 13.15 (br s, 1H), 9.58 (s, 1H), 9.39
(s, 1H), 8.90 (d, J = 7.5 Hz, 1H), 7.17 (d, J = 8.6 Hz, 2H), 7.13 (d, J =
8.6 Hz, 2H), 6.68 (d, J = 8.6 Hz, 2H), 5.28 (s, 1H), 5.08 (d, J = 8.5 Hz,
1H), 4.85 (ddd, J = 8.0, 5.3, 2.6 Hz, 1H), 3.34 (t, J = 5.3 Hz, 1H), 3.30
(dd, J = 4.8, 2.4 Hz, 1H), 1.37 (s, 9H). 13C NMR (101 MHz, DMSO-
d6): δ 171.2, 171.0, 166.4, 161.8, 157.9, 157.3, 129.6, 129.2, 128.9,
124.9, 116.0, 115.4, 82.1, 78.8, 57.4, 55.3, 47.3, 28.7. HRMS (FAB):
calcd for C24H28N3O8 486.18764, found 486.18742 [M + H]+.
N-(tert-Butyloxycarbonyl)nocardicin G (28b). The title compound
was prepared and purified analogously to compound 28a by replacing
27a with 27b (470 mg, 0.88 mmol). The product was obtained as a
light yellow solid (415 mg, 72%). [α]25 = −166.2 (c = 1.0, MeOH).
D
1H NMR (400 MHz; DMSO-d6): δ 13.15 (br s, 1H), 9.60 (s, 1H),
9.39 (s, 1H), 8.77 (d, J = 8.2 Hz, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.14
(d, J = 8.2 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H),
5.32 (s, 1H), 5.00 (d, J = 8.6 Hz, 1H), 4.85 (ddd, J = 7.7, 5.0, 2.4 Hz,
1H), 3.69 (t, J = 5.3 Hz, 1H), 2.89 (t, J = 2.2 Hz, 1H), 1.36 (s, 9H).
13C NMR (101 MHz, DMSO-d6): δ 170.8, 170.7, 166.1, 157.5, 156.9,
154.8, 129.5, 128.6, 128.4, 124.1, 115.5, 115.0, 78.3, 64.9, 56.9, 54.6,
46.4, 28.2. HRMS (FAB): calcd for C24H28N3O8 486.18764, found
486.18738 [M + H]+.
In a separate 50 mL pear-shaped flask was dissolved 25a (789 mg,
2.40 mmol) in 5 mL of reagent-grade DMF along with 2,6-lutidene
(370 μL, 3.26 mmol) and the mixture cooled to 0 °C in an ice bath.
The solution was added dropwise over 2 min to the mixture containing
epi-Nocardicin G-Pantetheine (29a). In a 10 mL round-bottomed
flask equipped with a magnetic stir bar was dissolved 28a (49 mg, 0.10
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mmol) in 2 mL of reagent-grade DMF, to this solution was added
K2CO3 (42 mg, 0.30 mmol), and the mixture was cooled to 0 °C with
an ice bath. To this cooled solution was added PyBOP (63 mg, 0.12
mmol) followed by 11 (39 mg, 0.12 mmol), and the reaction mixture
was stirred for 30 min at room temperature. The solution was diluted
with 40 mL of EtOAc and washed with satd aq NH4Cl (2 × 20 mL),
satd aq NaHCO3 (1 × 20 mL), and brine (1 × 20 mL). The EtOAc
was concentrated in vacuo to a viscous oil, which was redissolved in 2
mL of 70:30 ACN/H2O. The product was purified by prep method B,
collected, frozen on dry ice, and lyophilized to dryness. The
lyophilized powder was dissolved in 5 mL of reagent-grade TFA and
stirred at room temperature for 15 min. The TFA was removed in
vacuo, and the residue was resuspended in 2 mL of 70:30 H2O/ACN
supplemented with 0.1% TFA. The freshly deprotected product was
purified by prep method A, collected on dry ice, and lyophilized to
dryness to provide the product as a white TFA salt (32 mg, 41%).
Isolation of 2 mg of diasteromerically pure material was achieved using
analytical method B. 1H NMR (400 MHz; D2O): δ 7.26 (d, J = 8.7 Hz,
2H), 7.16 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.5
Hz, 2H), 5.54 (s, 1H), 5.02 (s, 1H), 4.76 (dd, J = 7.6, 2.1 Hz, 1H),
3.86 (s, 1H), 3.43 (t, J = 5.4 Hz, 1H), 3.40−3.27 (m, 5H), 3.39 (d, J =
11.2 Hz, 1H), 3.28 (d, J = 11.2 Hz, 1H), 3.02 (app td, J = 6.3, 2.5 Hz,
2H), 2.28−2.24 (m, 2H), 0.78 (s, 3H), 0.74 (s, 3H). 13C NMR (101
MHz, D2O): δ 199.3, 175.0, 173.9, 168.1, 162.8, 157.3, 156.6, 130.6,
130.6, 130.0, 123.9, 123.3, 116.4, 116.0, 75.7, 68.4, 64.9, 54.8, 47.5,
38.6, 38.3, 38.3, 35.3, 35.2, 28.5, 28.5, 20.5, 19.1. HRMS (FAB): calcd
for C30H40N5O9S 646.25468, found 646.25354 [M + H]+.
Nocardicin G-Pantetheine (29b). The title compound was
prepared and purified analogously to compound 29a by replacing
28a with 28b (49 mg, 0.10 mmol). The product was obtained as a
TFA salt (34 mg, 44%). Isolation of 2 mg of diasteromerically pure
material was achieved using analytical method B. 1H NMR (400 MHz;
D2O): δ 7.21 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H), 6.84 (2d, J
= 8.5 Hz, 4H), 5.56 (s, 1H), 4.96 (s, 1H), 4.80 (dd, J = 4.4, 1.3 Hz,
1H), 3.84 (s, 1H), 3.71 (t, J = 5.6 Hz, 1H), 3.37 (d, J = 11.2 Hz, 1H),
3.33−3.24 (m, 4H), 3.27 (d, J = 11.2 Hz, 1H), 3.09 (dd, J = 5.8, 2.4
Hz, 1H), 3.02 (t, J = 6.0 Hz, 3H), 2.26 (td, J = 6.4, 2.3 Hz, 2H), 0.78
(s, 3H), 0.73 (s, 3H). 13C NMR (101 MHz, D2O): δ 199.8, 175.0,
174.0, 169.1, 168.8, 157.3, 156.7, 130.8, 130.0, 123.8, 123.3, 116.4,
116.0, 75.7, 68.4, 65.0, 56.0, 54.6, 47.0, 38.6, 38.3, 35.4, 35.2, 28.5,
20.5, 19.1. HRMS (FAB): calcd for C30H40N5O9S 646.25468, found
646.25398 [M + H]+.
epi-Nocardicin G-SNAC (30a). In a 10 mL round-bottomed flask
equipped with a magnetic stir bar was dissolved 28a (276 mg, 0.57
mmol) in 2 mL of reagent-grade DMF, to this solution was added
DIEA (297 μL, 1.71 mmol), and the solution was cooled to 0 °C in an
ice bath. To this cooled solution was added PyBOP (325 mg, 0.63
mmol) followed by SNAC (63 μL, 0.63 mmol), and the reaction was
stirred for 30 min at room temperature. The reaction mixture was
diluted with 40 mL of EtOAc and washed with satd aq NH4Cl (2 × 20
mL), satd aq NaHCO3 (1 × 20 mL), and brine (1 × 20 mL). The
EtOAc was concentrated in vacuo to a viscous oil, which was
redissolved in 2 mL 70:30 ACN/H2O. The reaction was purified by
prep method A, and the product was collected, frozen on dry ice, and
lyophilized to dryness. The lyophilized powder was dissolved in 5 mL
of reagent-grade TFA and stirred at room temperature for 15 min. The
TFA was removed in vacuo, and the residue was resuspended in 2 mL
of 70:30 H2O/ACN supplemented with 0.1% TFA. The freshly
deprotected product was purified by prep method A, collected, frozen
on dry ice, and lyophilized to dryness to provide the product as a white
TFA salt (177 mg, 52%). Isolation of 2 mg of diasteromerically pure
material was achieved using analytical method B. 1H NMR (400 MHz;
D2O): δ 7.27 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 6.88 (d, J =
8.7 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.54 (s, 1H), 5.03 (s, 1H), 4.77
(dd, J = 5.2, 2.6 Hz, 1H), 3.42 (t, J = 5.6 Hz, 1H), 3.36 (dd, J = 6.0, 2.4
Hz, 1H), 3.26 (td, J = 6.2, 1.7 Hz, 2H), 3.01 (td, J = 6.2, 1.8 Hz, 2H),
1.76 (s, 3H). 13C NMR (101 MHz, D2O): δ 199.4, 174.2, 169.1, 168.1,
157.3, 156.6, 130.5, 130.0, 123.9, 123.4, 116.4, 116.0, 64.9, 56.0, 54.8,
47.5, 38.3, 28.4, 21.8. HRMS (ESI) calcd for C23H27N4O6S 487.16458,
found 487.1641 [M + H]+.
Nocardicin G-SNAC (30b). The title compound was prepared and
purified analogously to compound 30a by replacing 28a with 28b (276
mg, 0.57 mmol). The product was obtained as a white TFA salt (187
mg, 55%). Isolation of 2 mg of diasteromerically pure material was
1
achieved using analytical method B. H NMR (400 MHz; D2O): δ
7.21 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 6.84 (d, J = 8.7 Hz,
2H), 6.84 (d, J = 8.7 Hz, 2H), 5.55 (s, 1H), 4.96 (s, 1H), 4.80 (dd, J =
5.3, 2.6 Hz, 1H), 3.70 (t, J = 5.6 Hz, 1H), 3.27 (td, J = 6.1, 3.4 Hz,
2H), 3.08 (dd, J = 5.9, 2.6 Hz, 1H), 3.02 (t, J = 6.0 Hz, 2H), 1.77 (s,
3H). 13C NMR (101 MHz, D2O): δ 199.8, 174.2, 169.1, 168.8, 157.3,
156.6, 130.7, 130.0, 123.8, 123.2, 116.3, 116.0, 64.9, 56.0, 54.5, 47.0,
38.3, 28.4, 21.7. HRMS (ESI): calcd for C23H27N4O6S 487.16458,
found 487.1641 [M + H]+.
L-(p-Hydroxyphenyl)glycine-L-arginine-epi-nocardicin G-SNAC
(31a). In a 100 mL round-bottomed flask equipped with a magnetic
stir bar was dissolved compound 15 (281 mg, 0.37 mmol) in 10 mL of
reagent-grade THF. To this solution was added a catalytic amount of
Pd-OH/C, and the dipeptide was hydrogenated under 1 atm of H2 at
room temperature for 12 h. The mixture was filtered through Celite
and washed with THF (3 × 50 mL), and the filtrate was concentrated
in vacuo and used without further purification.
In a separate 10 mL round-bottomed flask equipped with a
magnetic stir bar was dissolved a mixture of 30a and 30b (200 mg,
0.33 mmol) in 5 mL of reagent-grade DMF containing K2CO3 (92 mg,
0.67 mmol) and the mxiture was cooled to 0 °C in an ice bath. In a
separate 10 mL round- bottomed flask was dissolved freshly
deprotected 15 in 2 mL of reagent-grade DMF, to this was added
K2CO3 (46 mg, 0.33 mmol), and the soltuion cooled to 0 °C with an
ice bath. Once both solutions were sufficiently cooled, PyBOP (160
mg, 0.37 mmol) was added to the flask containing the carboxylic acid,
and after 1 min, the solution containing 30a and 30b was transferred
dropwise over 2 min to the activated acid. The reaction mixture was
stirred at 0 °C for 30 min then at room temperature for 2 h. The
solution was diluted with 40 mL of EtOAc and washed with satd aq
NH4Cl (2 × 20 mL), NaHCO3 (1 × 20 mL) and brine (1 × 20 mL).
The EtOAc was concentrated in vacuo to a viscous oil which was
redissolved in 2 mL of 70:30 ACN/H2O. The reaction mixture was
purified by prep method B, and the product was collected, frozen on
dry ice, and lyophilized to dryness. The lyophilized product was
dissolved in 5 mL of reagent-grade TFA and stirred at room
temperature for 15 min. The TFA was removed in vacuo, and the
residue was resuspended in 2 mL of 70:30 H2O/ACN supplemented
with 0.1% TFA. The freshly deprotected product was purified by prep
method A to provide the product as a white TFA salt (2 mg, 7%).
Isolation of 1 mg of diasteromerically pure material was achieved using
analytical method A. 1H NMR (601 MHz; D2O): δ 7.19 (d, J = 8.6 Hz,
2H), 7.18 (d, J = 8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6
Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 5.55 (s,
1H), 5.23 (s, 1H), 5.00 (s, 1H), 4.28 (t, J = 7.3 Hz, 1H), 3.46−3.44
(m, 1H) 3.45, 3.42−3.40 (m, 1H), 3.29−3.26 (m, 2H), 3.05−3.01 (m,
4H), 1.76 (s, 3H), 1.69−1.67 (m, 2H), 1.51−1.42 (m, 1H), 1.41−1.35
(m, 1H). HRMS (FAB): calcd for C37H46N9O9S 792.31392, found
792.31368 [M + H]+.
L-(p-Hydroxyphenyl)glycine-L-arginine-nocardicin G-SNAC (31b).
The title compound was prepared and purified analogously to
compound 31a. The product was obtained as a white TFA salt (3
mg, 10%). Isolation of 1 mg of diasteromerically pure material was
1
achieved using analytical method B. H NMR (601 MHz; D2O): δ
7.18 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.1 Hz, 2H), 7.05 (d, J = 8.4 Hz,
2H), 6.84 (d, J = 8.6 Hz, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6
Hz, 2H), 5.58 (s, 1H), 5.14 (s, 1H), 4.97 (s, 1H), 4.23 (t, J = 7.4 Hz,
1H), 3.74 (t, J = 5.4 Hz, 1H), 3.33−3.27 (m, 2H), 3.15 (dd, J = 5.1, 2.0
Hz, 1H), 3.07−3.02 (m, 2H), 2.99 (app td, J = 7.0, 3.0 Hz, 2H), 1.80
(s, 3H), 1.65−1.60 (m, 2H), 1.45−1.39 (m, 1H), 1.36−1.31 (m, 1H).
HRMS (FAB): calcd for C37H46N9O9S 792.31392, found 792.31423
[M + H]+.
Rate Determination of Spontaneous Epimerization of Noc
G-SNAC to epi-Noc G-SNAC. To measure the rate of spontaneous
chemical epimerization of Noc G-SNAC 30b to epi-Noc G-SNAC 30a
at a biologically relevant pH, 500 μL of 1.0 mM Noc G-SNAC in 50
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mM KiPO4, pH 7.5 were prepared. Twenty-five microliters of the
reaction solution was aliquoted into 15 μL of a 0.2% TFA aqueous
solution on ice at t = 1, 2, 5, 10, 15, 20, 30, 45, 65, 80, 120, and 180
min. Reactions were repeated in triplicate. Quenched reactions were
analyzed via HPLC using analytical method B, monitoring absorption
at 272 nm. The rate of epimerization (k1 + k2) was calculated by fitting
the data to a nonlinear one-phase decay curve.
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ASSOCIATED CONTENT
■
(23) Roach, P. L.; Clifton, I. J.; Hensgens, C. M.; Shibata, N.;
Schofield, C. J.; Hajdu, J.; Baldwin, J. E. Nature 1997, 387, 827.
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S
* Supporting Information
1
Copies of the H, 13C, and 31P NMR spectra of all new
̈
̈
compounds. HPLC traces of thioesters with and without
incubation in 50 mM KiPO4, pH 7.5. This material is available
free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: ctownsend@jhu.edu. Tel/fax: (410) 516-7444/(410)
261-1233.
Notes
The authors declare no competing financial interest.
(31) El-Faham, A.; Funosas, R. S. s.; Prohens, R.; Albericio, F.
Chem.Eur. J. 2009, 15, 9404.
ACKNOWLEDGMENTS
■
(32) Valeur, E.; Bradley, M. Chem. Soc. Rev. 2009, 38, 606.
(33) Long, A. A. W.; Nayler, J. H. C.; Smith, H.; Taylor, T.; Ward, N.
J. J. Chem. Soc. 1971, 1920.
We warmly thank Dr. K. A. Moshos for chemical advice, helpful
discussions, and encouragement and Dr. I. P. Mortimer and Dr.
K. Belecki for all HRMS data reported. We are deeply grateful
to Dr. A. Majumdar and Dr. C. Moore for their NMR expertise,
guidance, and conducting of 400 and 600 MHz cryoprobe
experiments, and to the National Institutes of Health research
grant AI014937.
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