Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin Biosynthesis by Incorporation of Diverse d -Tyr Analogues

Article abstract of DOI:10.1021/acs.joc.8b00916

We report a general method for synthesizing diverse d-Tyr analogues, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogues were incorporated into the vancomycin substrate-peptide and reacted with

Full text of DOI:10.1021/acs.joc.8b00916

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Note
Modulating OxyB-Catalyzed Cross-Coupling Reactions in
Vancomycin Biosynthesis by Incorporation of Diverse D-Tyr Analogs
Seyma Ozturk, Clarissa C. Forneris, Andy K. L. Nguy, Erik J Sorensen, and Mohammad R. Seyedsayamdost
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00916 • Publication Date (Web): 28 May 2018
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Modulating OxyBꢀCatalyzed CrossꢀCoupling Reactions in Vancomycin
Biosynthesis by Incorporation of Diverse DꢀTyr Analogs
Seyma Ozturk,† Clarissa C. Forneris,† Andy K. L. Nguy,† Erik J. Sorensen,†,* and
Mohammad R. Seyedsayamdost†,‡,*
† Department of Chemistry, Princeton University, Princeton, NJ 08544
‡ Department of Molecular Biology, Princeton University, Princeton, NJ 08544
*Corresponding author. Eꢀmail addresses:
ejs@princeton.edu (E.J. Sorensen); mrseyed@princeton.edu (M.R. Seyedsayamdost)
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Abstract
We report a general method for synthesizing diverse
DꢀTyr analogs, one of the constituents of
the antibiotic vancomycin, using a Negishi crossꢀcoupling protocol. Several analogs were
incorporated into the vancomycin substrateꢀpeptide and reacted with the biosynthetic enzymes
OxyB and OxyA, which install the characteristic aromatic crosslinks. We find that even small
structural perturbations are not accepted by OxyA. The same modifications, however, enhance
the catalytic capabilities of OxyB leading to the formation of a new macrocycle within the
vancomycin framework.
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The discovery and development of penicillin has saved countless lives. Because
antibiotic therapy inevitably selects for resistance, there is a perpetual demand for the
deployment of new liveꢀsaving drugs.1 The need for antibiotics can be met in several ways,
including discovery of new antibiotic scaffolds or the alteration of existing ones that restore
efficacy against resistant pathogens. The latter strategy has been extremely successful and
numerous antibiotic now have second, third, and fourthꢀgeneration semiꢀsynthetic
derivatives.1a,2 The scope of this kind of derivatization is limited for the drug of last resort,
vancomycin (1, Fig. 1), because its multifunctional structure besets efforts to synthesize diverse,
siteꢀspecifically modified analogs.3,4 Though impressive total syntheses of the drug and useful
analogs have been reported, their preparations are still challenging.3b,5 We recently established
a new method for generating vancomycin derivatives using a combination of solidꢀphase
peptide synthesis (SPPS) and biocatalytic transformations.6 By using the native biosynthetic
metalloenzymes, we implemented a simple route in which the 7mer linear peptide is created by
SPPS and the synthetically challenging aryl crosslinks are installed enzymatically using the
cytochrome P450 enzymes OxyB, OxyA, and OxyC (Fig. 1).6 The creation of libraries of
vancomycin derivatives requires development of new methods for the synthesis of the
component amino acids, which may then be incorporated chemoꢀenzymatically into the final
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product. Herein, we report a method for synthesizing
reactions and demonstrate the preparation of derivatives containing a variety of substituents.
Incorporation of five ꢀTyr analogs at residue 6 of the vancomycin precursor peptide followed by
DꢀTyr using Negishi crossꢀcoupling
D
reaction with OxyB and OxyA revealed two crosslinks, involving rings CꢀD and, surprisingly,
rings AꢀB, rather than DꢀE (Fig. 1). Further assays revealed that OxyB installed both, while
OxyA did not react with the substrate variants. Thus, substitution of residue 6 appears to alter
the specificity of both enzymes, abolishing the reactivity of OxyA, but enhancing the catalytic
capabilities of OxyB.
Aside from
dihydroxyꢀ ꢀphenylglycine (
phenylglycine ( ꢀHpg), βꢀhydroxyꢀ3ꢀchloroꢀ
prepare vancomycin derivatives, synthetic access to analogs of each amino acid is essential.
For some amino acids such as ꢀTyr, a plethora of derivatives are commercially available and
enzymatic syntheses of variants have been reported as well.8 For others, however, notably
aromatic ꢀamino acids, new synthetic protocols are required. Given that OxyA crosslinks 3ꢀClꢀ
ꢀTyr at residue 6 of vancomycin, we focused our efforts on the synthesis of ꢀTyr variants.
L
ꢀAsn, vancomycin contains six nonꢀproteinogenic amino acids: 3,5ꢀ
ꢀDpg), βꢀhydroxyꢀ3ꢀchloroꢀ ꢀTyr (βꢀOHꢀ3ꢀClꢀ ꢀTyr), 4ꢀhydroxyꢀ
ꢀTyr (βꢀOHꢀ3ꢀClꢀ ꢀTyr), and Nꢀmethylꢀ
ꢀLeu.7 To
L
L
L
L
Dꢀ
D
D
D
D
L
D
D
D
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Figure 1. Structure of vancomycin. Bonds formed by OxyB, A, and C are highlighted, along with the ring
nomenclature.
DꢀTyr can be halogenated at the phenolic 3ꢀ and 5ꢀpositions by electrophilic aromatic
substitution (EAS). But introduction of the halogen typically requires harsh conditions and the
reaction scope is limited. Beyond halogenation and other basic modifications, methods for
introducing a wide array of substituents have not been reported. To that end, we envisioned a
Negishi crossꢀcoupling scheme between the organozinc derivative of protected 3ꢀiodoꢀ
Fig. 2a) with aryl halides containing the desired functionalities to convergently build
analogs.9 Using the protocol described by Jackson and coworkers, we began by crossꢀcoupling
the organozinc derivative of NꢀFmocꢀ3ꢀiodoꢀ ꢀAla methyl ester with readily available 4ꢀbromoꢀ2ꢀ
R phenols (R = OCH3, CH3, F, CF3) through the combined action of Pd2dba3 and P(oꢀtolyl)3 in
DMF (Fig. 2a).10 The NꢀFmocꢀ
ꢀTyr methyl esters thus obtained were then hydrolyzed using
LiOH in THF/water. This method effectively afforded a variety of NꢀFmocꢀprotected ꢀTyr
derivatives, ready for use in SPPS (3ꢀ6, Fig. 2b, Figs. S1ꢀS10). Similarly, NꢀFmocꢀ3,4ꢀ
dihydroxyꢀ ꢀPhe (NꢀFmocꢀ ꢀDOPA) was synthesized by Negishi coupling of 2 with 4ꢀbromoꢀ1,2ꢀ
dimethoxybenzene, followed by demethylation (7, Fig. 2b, Figs. S11ꢀ13). This approach also
proved convenient for generating protected 3ꢀchloroꢀ4ꢀaminoꢀ ꢀPhe (8, Fig. 2b, Figs. S14ꢀS15).
On the other hand, NꢀFmocꢀ3ꢀBrꢀ ꢀTyr and NꢀFmocꢀ3,5ꢀCl2ꢀ ꢀTyr were synthesized by EAS
using traditional methods (9, 10, Fig. 2c, Figs. S16ꢀS17). The combination of these procedures
enabled the creation of a small library of diverse ꢀTyr amino acids (Fig. 2).
With the ꢀTyr analogs in hand, we next established their use in SPPS, in which the Nꢀ
protected amino acids were coupled onto a hydrazinated 2ꢀchlorotrityl chloride resin using
Fmocꢀchemistry (Fig. 3a). Of the ꢀTyr analogs shown (Figs. 2b, 2c), we focused on those with
DꢀAla (2,
DꢀTyr
D
D
D
D
D
D
D
D
D
D
D
ortho substituents to examine their reactivity with OxyB and OxyA. Amino acids 3ꢀ5, 9, and 10
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Figure 2. Synthesis of
D
ꢀTyr analogs. (a) Negishi coupling scheme for the convergent synthesis of
D
ꢀTyr building
blocks. (b)
DꢀTyr derivatives synthesized using Pdꢀcatalyzed crossꢀcoupling. (c) Synthesis of halogenated DꢀTyr
residues by electrophilic aromatic substitution. Fmoc = fluorenylmethyloxycarbonyl; dba = dibenzylideneacetone;
DMF = N,Nꢀdimethylformamide; FmocOSu = 9ꢀfluorenylmethyl succinimidyl carbonate.
were incorporated into simplified 7mer peptides. As controls, we also inserted
Tyr at residue 6, both of which we employed previously to recapitulate the reaction of OxyA in
vitro.11 Aside from the
ꢀTyr analogs at residue 6, two additional changes were made relative to
the native vancomycin 7mer peptide: (1) we used 3ꢀClꢀ ꢀTyr at residue 2, rather than βꢀOHꢀ3ꢀ
Clꢀ ꢀTyr, as the latter repeatedly resulted in dehydration of the βꢀOH moiety; and (2) we chose
ꢀTyr at residue 1, rather than ꢀDpg, which has been reported to result in racemization during
DꢀTyr and 3ꢀClꢀDꢀ
D
L
L
L
L
SPPS. Each 7mer peptide was released as an acylꢀhydrazide (11ꢀ16, Fig. 3b, Figs. S18ꢀS22)
and subsequently converted into a coenzyme A (CoA) conjugate using a native chemical
ligationꢀbased strategy to deliver substrates 17ꢀ23 in good yields (Fig. 3c, Figs. S23ꢀ27).11
Compared to other methods, this approach has the advantage of mild being carried out in
aqueous solution at room temperature and neutral pH, which avoids epimerization of sensitive
residues.13 Together, these steps provided a facile procedure for synthesizing a variety of
thioesterified biosynthetic precursors containing natural and unnatural DꢀTyr building blocks.
Upon purification, the 7merꢀCoA adducts were used to attach the peptide substrate to an
Xꢀdomain/peptidylꢀcarrier protein (XꢀPCP) diꢀdomain. In this construct, the PCP domain carries
the phosphopantetheineꢀ7mer on a conserved active site Ser residue, while the Xꢀdomain
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recruits the cytochrome P450 enzymes to facilitate efficient crossꢀcoupling.14 Each substrate
was subsequently reacted with OxyB alone, OxyA alone, or OxyB and OxyA. At defined
timepoints, the reaction was quenched by release of the peptide from the XꢀPCP via
propylamineꢀmediated aminolysis, because amidation of the Cꢀterminus has been shown to
enhance the reactivity of vancomycin.5a,15 The products of the reaction were subsequently
assessed by HPLCꢀQtofꢀMS.
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Figure 3. Incorporation of
containing ꢀTyr variants to give acyl hydrazides. (b) 7mer peptide hydrazides described in this work. (c) Scheme for
synthesis of CoAꢀadducts of 7mer peptides. DIPEA = N,Nꢀdiisopropylethylamine; COMU = (1ꢀcyanoꢀ2ꢀethoxyꢀ2ꢀ
DꢀTyr analogs into vancomycin substrate peptides. (a) SPPS of vancomycin analogs
D
oxoethylidenaminooxy)dimethylaminoꢀmorpholinoꢀcarbenium
diazabicyclo[5.4.0]undecꢀ7ꢀene; TIS = triisopropylsilane.
hexafluorophosphate;
DBU
=
1,8ꢀ
Analysis of the reaction with
the CꢀOꢀD aryl ether bond, as previously established.11 Conversion to the singlyꢀcrosslinked
product proceeded in 86% yield. On the other hand, peptide 22 containing 3ꢀClꢀ ꢀTyr gave both
DꢀTyr at residue 6 (23) with OxyB and OxyA resulted only in
D
CꢀOꢀD and DꢀOꢀE crosslinks, as confirmed by HRꢀMSn analysis. The doublyꢀcrosslinked product
was formed in 20% yield. We have previously interpreted these results as indicative of the strict
substrate preference of OxyA toward a chlorinated residue 6.11
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With these benchmarks in place, we next examined the reactions of variants 17ꢀ21 (Fig.
4a). Incubation with OxyB and OxyA gave both singly and doublyꢀcrosslinked products as
inferred from a loss of 2 Da or 4 Da relative to each substrate, which was determined by highꢀ
resolution MS (HRꢀMS, Table S1). Interestingly, the same outcome was observed with OxyB
alone, whereas no reaction occurred at all when each substrate was incubated with OxyA only,
suggesting that OxyB installed both crosslinks. The products formed by OxyB were next
examined by HRꢀtandemꢀMS (HRꢀMS/MS) to assess the site of crosslink formation. For all
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substrates containing
DꢀTyr derivatives, we could clearly see nearly all b and y ions before
reaction with OxyB. After the OxyB reaction, however, the b5, b4, y3, and y4 ions were no
longer observed for the singlyꢀcrosslinked products (Fig. 4b). Moreover, fragments b7, b6, y5,
y6, y7 were now 2 Da lighter than the corresponding substrate fragments. These data are
consistent with installation of the canonical CꢀOꢀD aryl ether bond by OxyB (24, Fig. 4b, Figs.
S28ꢀ33, Tables S1ꢀS6).
Examination of the doublyꢀcrosslinked products also revealed the CꢀOꢀD linkage. Further
analysis, however, showed disappearance of b6 and b7 ions, which were clearly detectable in
the monocyclized product (Fig. 4b, 4c). Moreover, ions y5, y6, and y7 were now 4 Da lighter
than the corresponding fragments in the substrate and 2 Da lighter than those fragments in the
singlyꢀcyclized product. These results point to the presence of two crosslinks involving the first
four residues of the peptides, one consisting of a CꢀOꢀD link and the other involving rings A and
B (Fig. 4c, Figs. S28ꢀ33, Tables S1ꢀS6).
OxyB and its homologs from other glycopeptide biosynthetic gene clusters have been
shown to install aryl ether, rather than biaryl linkages.16 We therefore inferred that the unusual
AꢀB crosslink in peptides 17ꢀ21 would also be a bisꢀaryl ether linkage and could consist either of
the Hpg phenolꢀoxygen bound to the meta position of the Tyr side chain (25, BꢀOꢀA, Fig. 4c), or
the Tyrꢀphenolꢀoxygen linked to the meta position of Hpg (26, AꢀOꢀB, Fig. 4C). We utilized an
isotope replacement approach using 3,5ꢀ2H2ꢀ
(Fig. 4d). Reaction of OxyB with peptide 20, now bearing 3,5ꢀ2H2ꢀ
L
ꢀTyr to distinguish between these possibilities
ꢀTyr at residue 1 and 3ꢀBrꢀDꢀ
L
Tyr at residue 6, followed by analysis by HRꢀMSn revealed a ꢀ4 Da product peak, consistent with
the loss of four hydrogen atoms only and no loss of deuterons, thus suggesting formation of an
AꢀOꢀB, rather than BꢀOꢀA, macrocycle (Table S1, Fig. 4d). By extension, we propose that
products 27ꢀ31 also contain CꢀOꢀD and AꢀOꢀB macrocycles (Fig. 4d).
Finally, we quantified product yields with each of the DꢀTyr variants using HRꢀMS (Table
1). We note that each crosslink reduces the number of ionizable heteroatoms, thereby making
electrospray ionization progressively less efficient with CꢀOꢀD and AꢀOꢀBꢀbearing modifications.
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Figure 4. Formation of two crosslinks catalyzed by OxyB. (a) Attachment of CoAꢀadducts of 7mer peptides
substrates to the PCPꢀX diꢀdomain and summary of results in enzymatic assays with OxyA and OxyB, OxyB only, or
OxyA only. (b) Structure of monocyclized products catalyzed by OxyB, along with fragments observed by HRꢀMS/MS.
(c) Two possible structures of doublyꢀcrosslinked peptide products formed by OxyB, with HRꢀMS/MS fragments
shown. (d) Incorporation of 3,5ꢀ2H2ꢀTyr as a Cꢀterminal residue in a 7mer peptide carrying 3ꢀBrꢀ
DꢀTyr at residue 6.
Results of enzymatic assays reveal formation of an AꢀOꢀB crosslink in this variant and, by extension, in other residue
6 derivatives.
As such, MS quantification tends to underestimate the amounts of singlyꢀ and doublyꢀ
crosslinked products relative to substrate. With this caveat in mind, certain trends can
nonetheless be inferred from the data. Substitutions at the ortho position of
DꢀTyr lowers the
yields of CꢀOꢀD crosslink formation. The yields show a weak, but discernible, inverse correlation
to the van der Waals radii of each substituent, suggesting that steric effects, not so much
electronic ones, influence the OxyBꢀcatalyzed CꢀOꢀD yields. On the other hand, the yield of the
doublyꢀcrosslinked product containing the AꢀOꢀB bond did not show a clear correlation with
either electronic or steric properties for DꢀTyr at residue 6.
The results above have important implications for the generation of vancomycin
derivatives using our proposed chemoꢀenzymatic strategy. Even small modifications to residue
6 appear to render OxyA inactive, while the same modifications enhance the catalytic capability
of OxyB. Rather than a single crosslink, OxyB now installs CꢀOꢀD and AꢀOꢀB macrocycles, with
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the latter marking a new motif within the vancomycin framework. Thus new, doublyꢀcyclized
derivatives can be accessed with OxyB alone by altering the amino acids that occupy positions
6 and 1. The properties of the doublyꢀcyclized derivatives will be of interest in future studies, as
these analogs may exhibit new biological activities. Our results also suggest that vancomycin
derivatives, which maintain the cyclization pattern in the natural drug, will require an intact 3ꢀClꢀ
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D
ꢀTyr at residue 6.
Table 1. Product yields obtained upon reaction of vancomycin substrate peptides with OxyB.a
DꢀTyr
% CꢀOꢀD
% AꢀOꢀB
conversion
van der Waals
Radius (Å)
Analog
conversion
3ꢀH
3ꢀF
86%
60%
81%
17%
11%
16%
23%
0
3%
2%
0
1.2
1.47
1.52
1.75
1.75
1.85
2.0
3ꢀOMe
3ꢀCl
3,5ꢀCl2
3ꢀBr
4%
4%
10%
3ꢀMe
a Averages from two independent measurements are reported. Yields were determined by integrating the area under
the curve of each extracted ion chromatogram.
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Experimental Section
Materials and strains. All commercially available reagents and solvents were purchased from Ark
Pharm Inc., ChemꢀImpex International, Fisher Scientific, Oakwood Chemicals and Sigma Aldrich, and were
used directly without further purifications. Fmocꢀ and side chainꢀprotected amino acids, 2ꢀchlorotrityl
chloride resin and other components for solidꢀphase peptide synthesis were purchased from
Novabiochem/EMD Millipore and SigmaꢀAldrich. Reactions were monitored by thin layer chromatography
(TLC) carried out on 250 ꢁm Merck silica gel plates (60 F254) containing a fluorescent indicator (254 nm).
Visualization of the developed TLC plate was performed by irradiation with UV light. Organic solvents were
concentrated under reduced pressure on a Büchi rotary evaporator using a water bath (37°C). Flash
column chromatography was performed using Silicycle SiliaFlash P60 silica gel (60 Å pore size, 40 – 63
ꢁm particle size, 230 – 400 mesh).
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Amycolatopsis orientalis DSM40040 was obtained from the DSMZ. LB broth, Terrific Broth and
LB agar were purchased from Becton Dickinson. All antibiotics, IPTG, PMSF, lysozyme, βꢀ
mercaptoethanol, Sephadex Gꢀ25, COMU, NEt3, DIPEA, TIS, DBU, TFA, FmocꢀOSu, hydrazine
monohydrate, NaNO2, coenzyme A trilithium salt, glucoseꢀ6ꢀphosphate dehydrogenase, glucoseꢀ6ꢀ
phosphate, ferredoxin from spinach, ferredoxinꢀNADP+ reductase from spinach, and other components
necessary for biochemical assays were obtained from SigmaꢀAldrich. Nickel affinity resin and DNase I
were purchased from Clontech. Restriction enzymes, T4 DNA ligase, proofreading Q5 DNA polymerase,
and the corresponding buffers were purchased from New England Biolabs. PCR reactions were routinely
carried out in Failsafe buffer G (Epicentre).
1H NMR spectra were recorded on a Bruker 500 (500 MHz) and are referenced relative to
residual CHCl3 (in CDCl3) proton signal at δ 7.26 ppm or to residual acetonitrile (in CD3CN) proton signals
at δ 1.94 ppm. Data for 1H spectra are reported as follows: chemical shift (δ ppm), multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet, b = broad, ap = apparent), integration, coupling constant
(Hz) and assignment. 13C NMR spectra were recorded on a Bruker 500 (126 MHz) and are referenced
relative to residual CHCl3 (in CDCl3) at δ 77.16 ppm or to residual acetonitrile (in CD3CN) at δ 118.26 and
1.32 ppm. Data for 13C NMR spectra are reported in terms of chemical shift and multiplicity where
appropriate. IR spectra were recorded on a Nicolet 6700 FTꢀIR spectrometer with a 30000ꢀ200 cmꢀ1
diamond and are reported in terms of frequency of absorption (cmꢀ1). Highꢀresolution mass spectra
were obtained from Princeton University Mass Spectrometry Facility using an Agilent 6210 TOF LC/MS
(Electrospray Ionization). Optical rotations were measured on a Pelkin Elmer 341 Polarimeter at the
wavelength of 589 nm and a path length of 1.0 dm at 20°C.
N-Fmoc-D-serine methyl ester. DꢀSerine methyl ester hydrochloride (5.35 g, 34.4
CO2Me
HO
mmol) was dissolved in 100 mL water/dioxane (3:1) and NaHCO3 (7.22 g, 86.0 mmol)
NHFmoc
was added. The solution was cooled to 0 °C and FmocꢀOSu (11.83 g, 35.08 mmol) in
50 mL dioxane was added dropwise over 20 minutes. After complete addition, the suspension was stirred
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for 4 hours at room temperature. 40 mL water was added, and the solution was extracted into EtOAc (3 x
100 mL). The combined organic layers were washed with 1 N HCl (20 mL) and brine (40 mL), then dried
over anhydrous Na2SO4, and concentrated in vacuo. The resulting white solid was recrystallized from hot
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Et2O/hexanes. Filtration and washing with cold hexanes provided the product (11.10 g, 95%). H NMR
(500 MHz, CDCl3): δ 7.77 (d, J = 7.5 Hz, 2H), 7.61 (dd, J = 7.8, 3.3 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.33
(tt, J = 7.4, 1.4 Hz, 2H), 5.68 (d, J = 7.6 Hz, 1H), 4.45 (t, J = 8.5 Hz, 3H), 4.24 (t, J = 6.9 Hz, 1H), 4.01 (s,
1H), 3.95 (s, 1H), 3.81 (s, 3H), 2.05 (s, 1H). 13C NMR (126 MHz, CDCl3): δ 171.0, 156.3, 143.9, 143.8,
141.5, 127.9, 127.2, 125.2, 120.2, 67.3, 63.5, 56.1, 53.0, 47.3. MSꢀESI m/z 364.1159 ([M + Na]+,
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C19H19NNaO5, calc. 364.1161). [α]D = −3.7 (c 1.24, CH2Cl2). IR (neat): 3316, 1741, 1685, 1532, 1438,
1304, 1255.
N-Fmoc-3-iodo-D-alanine methyl ester (2). A 100ꢀmL flask was charged with
triphenylphosphine (3.28 g, 12.5 mmol) and imidazole (0.85 g, 12.5 mmol) and 40 mL
CH2Cl2, cooled to 0 °C in an ice bath, and I2 (3.17 g, 12.5 mmol) was added in three
portions. The mixture was warmed to room temperature and stirred for 20 min, and reꢀ
cooled to 0 °C. A solution of NꢀFmocꢀDꢀserine methyl ester (3.41 g, 10 mmol) in 10 mL CH2Cl2 was then
added dropwise, and the reaction mixture was stirred for 4 hours. It was then concentrated, and ether
was added to crash out the phosphine oxide. The mixture was filtered through celite with ether as the
eluent. The filtrate was then concentrated in vacuo, and the residue was purified on silica using CH2Cl2 to
yield the product as white solid (3.84 g, 85%). 1H NMR (500 MHz, CDCl3): δ 7.78 (d, J = 7.5 Hz, 2H), 7.62
(d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (tdd, J = 7.4, 3.1, 1.1 Hz, 2H), 5.67 (d, J = 7.6 Hz, 1H),
4.59 (dt, J = 7.6, 3.9 Hz, 1H), 4.44 (dd, J = 10.7, 7.4 Hz, 1H), 4.38 (dd, J = 10.6, 7.1 Hz, 1H), 4.26 (t, J =
7.3 Hz, 1H), 3.83 (s, 3H), 3.65 – 3.57 (m, 2H). 13C NMR (126 MHz, CDCl3): δ 169.9, 155.5, 143.9, 143.7,
141.5, 127.9, 127.3, 127.2, 125.3, 125.2, 120.2, 67.5, 54.2, 53.4, 47.2, 7.6. MSꢀESI m/z 474.0181 ([M +
Na]+, C19H18INNaO4, calc. 474.0178). [α]D 20 = −31.6 (c 0.71, CH2Cl2). IR (neat): 3313, 3053, 2944, 1736,
1724, 1691, 1537, 1446, 1313, 1278, 1226.
Preparation of D-tyrosine analogs: Standard procedure for cross-coupling reactions. Zinc dust
(190 mg, 3.0 mmol) was added to a flameꢀdried, nitrogenꢀpurged side arm roundꢀbottomed flask. Dry
DMF (1 mL) was added via syringe, followed by a catalytic amount of iodine (50 mg, 0.2 mmol). A color
change from colorless to yellow and back again was observed. NꢀFmocꢀ3ꢀiodoꢀDꢀalanine methyl ester (2)
(451 mg, 1.0 mmol) was added immediately, followed by a catalytic amount of iodine (50 mg, 0.2 mmol).
The solution was stirred at room temperature for 10 minutes and completion of zinc insertion was
observed by TLC. The reaction was accompanied by a noticeable exotherm. Pd2dba3 (45.8 mg, 0.05
mmol), P(oꢀtolyl)3 (60.8 mg, 0.20 mmol) and aryl bromide (1.3 equivalents relative to 2) were added to the
flask. The reaction was stirred for 10 minutes at room temperature and then at 50 °C for 6 hours under
positive pressure of nitrogen gas. The crude reaction mixture was applied directly to a silica gel column.
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Standard procedure for hydrolysis of tyrosine methyl esters. NꢀFmocꢀ3ꢀRꢀDꢀTyr methyl ester
(0.50 mmol) was dissolved in 5 mL THF/water (1:1) and the solution was cooled to 0 °C in an ice bath.
LiOH (0.75 mmol) was added and the reaction was stirred vigorously for 4ꢀ6 hours. The pH was then
adjusted to 2ꢀ3 with 1N HCl (aq.) solution, THF was removed in vacuo, and the aqueous layer was
extracted with EtOAc three times. The combined organic extracts were dried over anhydrous Na2SO4,
concentrated, and purified on silica using hexanes/EtOAc/AcOH (5:5:0.1) to afford NꢀFmocꢀRꢀDꢀTyr.
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N-Fmoc-3-methyl-D-tyrosine methyl ester. The compound was synthesized
Me
CO2Me
from 4ꢀBromoꢀ2ꢀmethylphenol using the standard crossꢀcoupling procedure and
purified by flash column chromatography using hexanes/EtOAc (4:1) as the
NHFmoc
HO
eluting solvent. Pure material was obtained as a white solid (310 mg, 72%). 1H NMR (500 MHz, CDCl3): δ
7.77 (dt, J = 7.6, 0.9 Hz, 2H), 7.57 (t, J = 8.5 Hz, 2H), 7.42 – 7.38 (m, 2H), 7.31 (tdd, J = 7.5, 2.3, 1.1 Hz,
2H), 6.86 (d, J = 2.1 Hz, 1H), 6.79 (dd, J = 8.0, 2.2 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H), 5.26 (d, J = 8.4 Hz,
1H), 4.82 (s, 1H), 4.63 (dt, J = 8.3, 5.8 Hz, 1H), 4.46 – 4.40 (m, 1H), 4.35 (dd, J = 10.7, 7.0 Hz, 1H), 4.22
(t, J = 7.1 Hz, 1H), 3.74 (s, 3H), 3.08 – 2.97 (m, 2H), 2.20 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 172.3,
155.8, 153.2, 144.0, 143.9, 141.4, 132.0, 128.0, 127.9, 127.8, 127.2, 125.3, 125.2, 124.1, 120.1, 115.2,
20
67.2, 55.1, 52.5, 47.3, 37.6, 15.9. MSꢀESI m/z 432.1807 ([M + H]+, C26H26NO5, calc. 432.1811). [α]D
=
−27.7 (c 0.22, CH2Cl2). IR (neat): 3312, 2951, 1726, 1694, 1512, 1448, 1266, 1215.
N-Fmoc-3-methyl-D-tyrosine (3). The compound was prepared using the
standard hydrolysis procedure. Pure material was obtained as a white solid (131
mg, 63%). 1H NMR (500 MHz, CD3CN): δ 7.82 (d, J = 7.6 Hz, 2H), 7.60 (dd, J =
7.7, 4.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.31 (q, J = 7.0 Hz, 2H), 6.96 (s, 1H),
6.88 (d, J = 8.2 Hz, 1H), 6.68 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 8.3 Hz, 1H), 4.36 – 4.21 (m, 3H), 4.18 (t, J =
7.0 Hz, 1H), 3.04 (dd, J = 14.1, 5.0 Hz, 1H), 2.80 (dd, J = 14.0, 9.0 Hz, 1H), 2.12 (s, 3H). 13C NMR (126
MHz, CD3CN): δ 173.4, 156.8, 154.7, 145.0, 144.9, 142.0, 132.6, 129.0, 128.6, 128.5, 128.0, 126.2,
126.1, 125.0, 120.9, 115.5, 67.2, 56.3, 47.9, 37.1, 16.1. [α]D 20 = −20.0 (c 0.55, CH2Cl2). MSꢀESI m/z
418.1652 ([M + H]+, C25H24NO5, calc. 418.1654). IR (neat): 3327, 2925, 1697, 1611, 1510, 1449, 1262,
1212.
N-Fmoc-3-methoxy-D-tyrosine methyl ester. The compound was synthesized
MeO
HO
CO2Me
from 4ꢀBromoꢀ2ꢀmethoxyphenol using the standard crossꢀcoupling procedure
NHFmoc
and purified by flash column chromatography using hexane/EtOAc (4:1) as
1
the eluting solvent. Pure material was isolated as a white solid (313 mg, 70%). H NMR (500 MHz,
CDCl3): δ 7.77 (d, J = 7.5 Hz, 2H), 7.56 (t, J = 8.5 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 7.5, 1.1
Hz, 2H), 6.83 (d, J = 8.0 Hz, 1H), 6.61 (d, J = 1.9 Hz, 1H), 6.58 (dd, J = 8.1, 1.9 Hz, 1H), 5.57 (s, 1H),
5.27 (d, J = 8.3 Hz, 1H), 4.64 (dt, J = 8.4, 5.9 Hz, 1H), 4.40 (dt, J = 6.8, 3.1 Hz, 2H), 4.21 (t, J = 7.1 Hz,
1H), 3.82 (s, 3H), 3.74 (s, 3H), 3.11 – 3.00 (m, 2H). 13C NMR (126 MHz, CDCl3): δ 172.2, 155.7, 146.6,
144.9, 144.0, 143.8, 141.4, 127.9, 127.6, 127.2, 125.2, 122.3, 120.2, 120.1, 114.6, 111.6, 67.1, 56.0,
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55.0, 52.5, 47.3, 38.1. MSꢀESI m/z 448.1755 ([M + H]+, C26H26NO6, calc. 448.1760). [α]D 20 = −26.4 (c
1.04, CH2Cl2). IR (neat): 3314, 1750, 1685, 1516, 1446, 1433, 1270, 1240, 1215.
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N-Fmoc-3-methoxy-D-tyrosine (4). The compound was prepared using the
standard hydrolysis procedure. Pure material was isolated as a white solid (136
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mg, 65%). 1H NMR (500 MHz, CD3CN): δ 7.82 (d, J = 7.6 Hz, 2H), 7.59 (d, J =
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7.6 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.30 (q, J = 7.0 Hz, 2H), 6.85 – 6.81 (m, 1H), 6.73 (d, J = 8.0 Hz,
1H), 6.69 – 6.64 (m, 1H), 5.95 (d, J = 8.5 Hz, 1H), 4.36 (td, J = 8.8, 4.8 Hz, 1H), 4.32 – 4.22 (m, 2H), 4.18
(t, J = 7.1 Hz, 1H), 3.79 (s, 3H), 3.09 (dd, J = 14.1, 4.9 Hz, 1H), 2.84 (dd, J = 14.1, 9.2 Hz, 1H). 13C NMR
(126 MHz, CD3CN): δ 173.5, 156.8, 147.9, 145.8, 145.0, 144.9, 142.0, 129.6, 128.6, 128.0, 126.1, 126.0,
122.7, 120.9, 115.4, 113.6, 67.2, 56.5, 56.2, 47.8, 37.5. [α]D 20 = −15.5 (c 1.24, CH2Cl2). MSꢀESI m/z
434.1604 ([M + H]+, C25H24NO6, calc. 434.1604). IR (neat): 3325, 2926, 1700, 1514, 1449, 1430, 1270,
1234, 1208.
F
CO2Me
N-Fmoc-3-fluoro-D-tyrosine methyl ester. The compound was synthesized from
4ꢀBromoꢀ2ꢀfluorophenol using the standard crossꢀcoupling procedure and
purified by flash column chromatography using hexane/EtOAc (4:1) as the
NHFmoc
HO
eluting solvent. Pure material was isolated as a white solid (195 mg, 45%). 1H NMR (500 MHz, CDCl3): δ
7.77 (d, J = 7.6 Hz, 2H), 7.57 (t, J = 7.1 Hz, 2H), 7.41 (td, J = 7.5, 2.3 Hz, 2H), 7.35 – 7.29 (m, 2H), 6.90
(t, J = 8.6 Hz, 1H), 6.82 (dd, J = 11.2, 2.0 Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 5.28 (d, J = 8.3 Hz, 1H), 5.25
(s, 1H), 4.63 (dt, J = 8.3, 5.7 Hz, 1H), 4.47 (dd, J = 10.7, 7.1 Hz, 1H), 4.37 (dd, J = 10.7, 6.8 Hz, 1H), 4.22
(t, J = 7.0 Hz, 1H), 3.74 (s, 3H), 3.03 (qd, J = 14.0, 5.7 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ 171.9,
155.7, 151.9, 150.0, 144.0, 143.8, 142.9, 142.7, 141.5, 128.63, 128.59, 127.9, 127.22, 127.20, 125.83,
125.81, 125.2, 125.1, 120.2, 117.5, 117.4, 116.5, 116.4, 67.1, 54.9, 52.6, 47.3, 37.5. 19F NMR (282 MHz,
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CDCl3): δ ꢀ140.25 (t, J = 10.3 Hz). MSꢀESI m/z 436.1563 ([M + H]+, C25H23FNO5, calc. 436.1560). [α]D
=
−27.1 (c 1.02, CH2Cl2). IR (neat): 3309, 2950, 1727, 1692, 1519, 1446, 1264, 1218.
N-Fmoc-3-fluoro-D-tyrosine (5). The compound was prepared using the
standard hydrolysis procedure. Pure material was isolated as a white solid (176
1
mg, 84%). H NMR (500 MHz, CD3CN): δ 7.83 (d, J = 7.7 Hz, 2H), 7.60 (d, J =
7.6 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.32 (td, J = 7.4, 3.8 Hz, 2H), 6.98 (d, J =
12.2 Hz, 1H), 6.86 (d, J = 4.6 Hz, 2H), 5.97 (d, J = 8.5 Hz, 1H), 4.36 (td, J = 8.6, 4.9 Hz, 1H), 4.30 (dd, J =
10.4, 7.4 Hz, 1H), 4.24 (t, J = 8.7 Hz, 1H), 4.18 (t, J = 7.0 Hz, 1H), 3.08 (dd, J = 14.3, 4.9 Hz, 1H), 2.85
(dd, J = 14.1, 9.1 Hz, 1H). 13C NMR (126 MHz, CD3CN): δ 173.2, 156.8, 152.8, 150.9, 145.0, 144.9,
144.0, 143.9, 142.0, 130.4, 130.3, 128.6, 128.0, 126.5, 126.4, 126.1, 126.0, 120.9, 118.39, 118.37, 117.7,
117.6, 67.3, 56.0, 47.9, 36.9. 19F NMR (282 MHz, CD3CN): δ ꢀ138.77 (d, J = 12.0 Hz). [α]D 20 = −26.0 (c
0.25, CH2Cl2). MSꢀESI m/z 422.1400 ([M + H]+, C24H21FNO5, calc. 422.1404). IR (neat): 3397, 3324,
3306, 2926, 2851, 1698, 1516, 1448, 1259.
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N-Fmoc-3-trifluoromethyl-D-tyrosine methyl ester. The compound was
F3C
HO
CO2Me
synthesized from 4ꢀBromoꢀ2ꢀ(trifluoromethyl)phenol using the standard crossꢀ
NHFmoc
coupling procedure and purified by flash column chromatography using
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hexane/EtOAc (4:1) as the eluting solvent. Pure material was isolated as a white solid (97 mg, 20%). H
NMR (500 MHz, CDCl3): δ 7.77 (d, J = 7.4 Hz, 2H), 7.57 (t, J = 6.6 Hz, 2H), 7.41 (dd, J = 8.6, 6.5 Hz, 2H),
7.32 (tt, J = 7.5, 1.6 Hz, 2H), 7.25 – 7.24 (m, 1H), 7.15 – 7.10 (m, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.80 (s,
1H), 5.33 – 5.27 (m, 1H), 4.64 (dt, J = 8.2, 5.7 Hz, 1H), 4.48 – 4.41 (m, 1H), 4.37 (dd, J = 10.7, 6.9 Hz,
1H), 4.21 (t, J = 6.9 Hz, 1H), 3.74 (s, 3H), 3.12 (dd, J = 14.0, 5.7 Hz, 1H), 3.05 (dd, J = 14.0, 5.9 Hz, 1H).
13C NMR (126 MHz, CDCl3): δ 171.8, 155.7, 152.93, 152.92, 143.9, 143.80, 143.77, 141.5, 134.5, 128.2,
127.9, 127.64, 127.62, 127.58, 127.55, 127.2, 125.2, 125.1, 120.2, 120.1, 118.1, 117.0, 116.7, 116.5,
116.2, 67.2, 54.9, 52.7, 47.3, 37.5. 19F NMR (282 MHz, CDCl3): δ ꢀ60.96. [α]D 20 = −28.3 (c 0.20, CH2Cl2).
MSꢀESI m/z 486.1532 ([M + H]+, C26H23F3NO5, calc. 486.1528). IR (neat): 3351, 2955, 2937, 2861, 1693,
1622, 1514, 1444, 1323, 1305, 1271.
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N-Fmoc-3-trifluoromethyl-D-tyrosine (6). The compound was prepared using the
standard hydrolysis procedure. Pure material was isolated as a white solid (169
mg, 72%). 1H NMR (500 MHz, CD3CN): δ 7.82 (d, J = 7.6 Hz, 2H), 7.59 (d, J =
7.5 Hz, 2H), 7.45 – 7.37 (m, 3H), 7.30 (dt, J = 15.0, 7.5 Hz, 3H), 6.92 (d, J = 8.4 Hz, 1H), 6.02 (d, J = 8.5
Hz, 1H), 4.38 (td, J = 8.8, 5.0 Hz, 1H), 4.26 (qd, J = 10.6, 7.4 Hz, 2H), 4.17 (t, J = 7.0 Hz, 1H), 3.14 (dd, J
= 14.2, 5.0 Hz, 1H), 2.90 (dd, J = 14.1, 9.2 Hz, 1H). 13C NMR (126 MHz, CD3CN): δ 173.2, 156.9, 154.71,
154.70, 145.0, 144.9, 142.03, 142.01, 135.4, 129.4, 128.62, 128.55, 128.51, 128.47, 128.4, 128.0,
126.13, 126.09, 126.06, 123.94, 120.90, 120.89, 117.8, 117.2, 117.0, 116.7, 116.5, 67.3, 56.0, 47.9, 36.8.
19F NMR (282 MHz, CD3CN): δ ꢀ62.65. MSꢀESI m/z 472.1370 ([M + H]+, C25H21F3NO5, calc. 472.1372).
[α]D 20 = −1.23 (c 1.14, CH3OH). IR (neat): 3303, 3068, 2924, 2853, 1694, 1622, 1513, 1446, 1323, 1305,
1269, 1202.
N-Fmoc-3,4-dimethoxy-D-phenylalanine methyl ester. This compound was
MeO
MeO
CO2Me
synthesized from 4ꢀbromoveratrole using the standard crossꢀcoupling
procedure. The crude reaction mixture was applied directly to a silica gel
NHFmoc
column, purified using hexane/EtOAc (5:1) as the eluting solvent. Pure material was isolated as a white
solid (336 mg, 73%). 1H NMR (500 MHz, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H), 7.56 (t, J = 8.5 Hz, 2H), 7.40
(t, J = 7.5 Hz, 2H), 7.34 – 7.28 (m, 2H), 6.78 (d, J = 7.9 Hz, 1H), 6.66 – 6.61 (m, 2H), 5.25 (d, J = 8.3 Hz,
1H), 4.66 (dt, J = 8.4, 5.9 Hz, 1H), 4.39 (d, J = 7.1 Hz, 2H), 4.21 (t, J = 7.2 Hz, 1H), 3.85 (s, 3H), 3.82 (s,
3H), 3.74 (s, 3H), 3.07 (t, J = 5.4 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ 172.2, 155.7, 149.0, 148.3,
144.0, 143.8, 141.43, 141.42, 128.2, 127.9, 127.2, 125.2, 121.6, 120.14, 120.13, 112.4, 111.3, 67.2, 56.0,
55.9, 55.0, 52.5, 47.3, 38.0. MSꢀESI m/z 462.1913 ([M + H]+, C27H28NO6, calc. 462.1917). [α]D 20 = −28.5
(c 0.42, CH2Cl2). IR (neat): 3322, 2926, 1752, 1693, 1535, 1517, 1447, 1267, 1236.
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N-Fmoc-D-DOPA (7). Dry dichloromethane (5 mL) and NꢀFmocꢀ3,4ꢀdimethoxyꢀ
Dꢀphenylalanine methyl ester (336 mg, 0.73 mmol) were added to a 25ꢀmL
6
flameꢀdried flask under inert atmosphere. The reaction mixture was cooled to ꢀ
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8
9
78°C in a cold bath using acetone/dry ice and boron tribromide (1.0 M in CH2Cl2, 2.26 mL, 2.26 mmol)
was added dropꢀwise. The cold bath was removed, and the mixture was stirred for 30 min, poured into ice
water, stirred for 30 min, saturated with salt, and extracted with dichloromethane three times. The
combined organic extracts were dried and concentrated in vacuo. The crude mixture was purified by flash
column chromatography using hexane/EtOAc (3:1) as the eluting solvent to afford NꢀFmocꢀDꢀDOPA
methyl ester (101 mg, 32%). Then eluting solvent was changed to hexane/EtOAc/AcOH (2:2:0.05) to
obtain NꢀFmocꢀDꢀDOPA (156 mg, 51%). Using the standard hydrolysis procedure, NꢀFmocꢀDꢀDOPA
methyl ester was converted into NꢀFmocꢀDꢀDOPA as well. NꢀFmocꢀDꢀDOPA methyl ester: 1H NMR (500
MHz, CDCl3): δ 7.76 (d, J = 7.5 Hz, 2H), 7.55 (d, J = 7.6 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.5
Hz, 2H), 6.74 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 2.0 Hz, 1H), 6.50 (dd, J = 8.0, 2.0 Hz, 1H), 5.71 (s, 2H),
5.33 (d, J = 8.4 Hz, 1H), 4.61 (dt, J = 8.3, 6.0 Hz, 1H), 4.43 (dd, J = 10.7, 7.1 Hz, 1H), 4.35 (dd, J = 10.7,
6.8 Hz, 1H), 4.20 (t, J = 7.1 Hz, 1H), 3.72 (s, 3H), 3.01 (dd, J = 14.1, 5.7 Hz, 1H), 2.94 (dd, J = 14.0, 6.2
Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 172.3, 155.9, 143.9, 143.84, 143.79, 143.1, 141.5, 141.4, 128.4,
127.9, 127.3, 127.2, 125.2, 125.1, 121.8, 120.2, 120.1, 116.3, 115.6, 67.2, 55.1, 52.6, 47.2, 37.8. MSꢀESI
m/z 434.1602 ([M + H]+, C25H24NO6, calc. 434.1604). [α]D 20 = −23.4 (c 0.80, CH2Cl2). IR (neat): 3316,
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3052, 2951, 1737, 1726, 1691, 1530, 1447, 1343, 1269, 1219. NꢀFmocꢀDꢀDOPA: H NMR (500 MHz,
CD3CN): δ 7.82 (d, J = 7.7 Hz, 2H), 7.60 (dd, J = 7.6, 3.9 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.32 (td, J =
7.5, 4.2 Hz, 2H), 6.73 (d, J = 8.1 Hz, 2H), 6.58 (dd, J = 8.0, 2.1 Hz, 1H), 5.92 (d, J = 8.4 Hz, 1H), 4.30 (m,
3H), 4.19 (t, J = 7.1 Hz, 1H), 3.03 (dd, J = 14.1, 5.0 Hz, 1H), 2.80 (dd, J = 14.1, 9.1 Hz, 1H). 13C NMR
(126 MHz, CD3CN): δ 173.4, 156.9, 145.3, 145.0, 144.9, 144.2, 142.0, 130.0, 128.6, 128.1, 126.2, 126.1,
122.0, 120.9, 117.1, 116.1, 67.3, 56.2, 47.9, 37.2. MSꢀESI m/z 420.1445 ([M + H]+, C24H22NO6, calc.
420.1447). [α]D 20 = −17.4 (c 0.85, CH2Cl2). IR (neat): 3447, 3330, 2156, 2024, 1765, 1639, 1555, 1489,
1387, 1310.
N-Fmoc-3-chloro-4-amino-D-phenylalanine methyl ester. This compound was
Cl
CO2Me
synthesized from 2ꢀchloroꢀ4ꢀbromoaniline using the standard crossꢀcoupling
NHFmoc
procedure. The crude reaction mixture was applied directly to a silica gel
H2N
1
column, purified using hexane/EtOAc (4:1) as the eluting solvent. White solid (297 mg, 66%). H NMR
(500 MHz, CDCl3): δ 7.77 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 8.7 Hz, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.36 – 7.29
(m, 2H), 7.01 (d, J = 2.1 Hz, 1H), 6.79 (dd, J = 8.2, 2.0 Hz, 1H), 6.68 (d, J = 8.2 Hz, 1H), 5.27 (d, J = 8.3
Hz, 1H), 4.61 (dt, J = 8.3, 5.6 Hz, 1H), 4.45 (dd, J = 10.7, 7.4 Hz, 1H), 4.35 (dd, J = 10.6, 7.0 Hz, 1H),
4.22 (t, J = 7.2 Hz, 1H), 4.00 (s, 2H), 3.74 (s, 3H), 2.99 (q, J = 8.6, 7.1 Hz, 2H). 13C NMR (126 MHz,
CDCl3): δ 172.0, 155.7, 144.0, 143.9, 142.1, 141.4, 130.2, 128.7, 127.9, 127.23, 127.22, 126.4, 125.3,
125.2, 120.14, 120.13, 119.4, 116.11, 67.2, 54.9, 52.5, 47.3, 37.3. [α]D 20 = −18.1 (c 0.26, CH2Cl2). MSꢀ
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ESI m/z 451.1425 ([M + H]+, C25H24ClN2O4, calc. 451.1425). IR (neat): 3359, 2950, 1709, 1623, 1506,
1447, 1308, 1250, 1210.
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N-Fmoc-3-chloro-4-(tert-butoxycarbonyl)amino-D-phenylalanine (8). The
purified crossꢀcoupled NꢀFmocꢀ3ꢀchloroꢀ4ꢀaminoꢀDꢀphenylalanine methyl
9
ester product was hydrolyzed using the standard hydrolysis procedure. After
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workup, the crude product was dissolved in 10 ml dioxane/water. NaHCO3
(2.5 eq.) and Boc2O (2.0 eq.) was added to the solution. The reaction mixture was stirred overnight and
then extracted into EtOAc (3x10 ml). The combined extracts were dried over anhydrous Na2SO4 and
concentrated in vacuo. The crude mixture was purified by flash column chromatography using
hexane/EtOAc/AcOH (2:2:0.01) as the eluting solvent to afford the product (211 mg, 60% over 2 steps).
1H NMR (500 MHz, CD3CN): δ 7.86 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H),
7.40 (t, J = 7.5 Hz, 2H), 7.31 (ddd, J = 10.9, 6.0, 2.1 Hz, 3H), 7.16 – 7.10 (m, 2H), 6.01 (d, J = 8.5 Hz,
1H), 4.40 (td, J = 8.8, 4.8 Hz, 1H), 4.29 (dd, J = 10.5, 7.2 Hz, 1H), 4.23 (dd, J = 10.5, 7.0 Hz, 1H), 4.17 (t,
J = 6.9 Hz, 1H), 3.13 (dd, J = 14.1, 5.0 Hz, 1H), 2.93 – 2.85 (m, 1H), 1.49 (s, 9H). 13C NMR (126 MHz,
CD3CN): δ 173.0, 156.8, 153.6, 145.0, 144.9, 142.1, 142.0, 134.9, 134.4, 130.8, 129.5, 128.6, 128.1,
126.2, 126.1, 122.5, 120.92, 120.91, 81.4, 67.3, 55.7, 47.9, 36.9, 28.4. MSꢀESI m/z 537.1798 ([M + H]+,
C29H30ClN2O6, calc. 537.1792). [α
1449, 1227.
20 = −4.9 (c 2.14, CH3OH). IR (neat): 3420, 2919, 2849, 1711, 1520,
D
N-Fmoc-3-bromo-D-tyrosine (9). DꢀTyrosine (0.9 g, 4.97 mmol) was dissolved in
100 mL of 0.5 N HCl (aq.). A solution containing KBrO3 (0.28 g, 1.66 mmol) and
KBr (1.19 g, 9.94 mmol) in 100 mL of H2O was added dropwise. The reaction
1
mixture was stirred overnight at room temperature. H NMR spectrum of crude
reaction mixture showed near 100% bromination. The solution was concentrated and used for the next
step without further purification. The crude product was dissolved in 50 mL dioxane/water (1:1), and
NaHCO3 (1.0 g, 11.93 mmol) was added. The solution was cooled in an ice bath and FmocꢀOSu (1.8 g,
5.46 mmol) was added in portions. The reaction mixture was stirred overnight at room temperature. 25
mL water was added, and the solution was extracted into EtOAc (3 x 50 mL). The combined organic
layers were washed with brine (40 mL), then dried over anhydrous Na2SO4, and concentrated in vacuo.
1
The resulting white solid was recrystallized from hot Et2O/hexane (2.15 g, 90%). H NMR (500 MHz,
CDCl3): δ 7.77 (d, J = 7.4 Hz, 2H), 7.56 (t, J = 7.4 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (td, J = 7.4, 3.3
Hz, 2H), 7.27 (s, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 5.24 (d, J = 7.8 Hz, 1H), 4.65 (q, J
= 5.6 Hz, 1H), 4.48 (dd, J = 10.4, 7.3 Hz, 1H), 4.38 (dd, J = 10.4, 7.0 Hz, 1H), 4.21 (t, J = 6.7 Hz, 1H),
3.11 (dd, J = 14.1, 5.2 Hz, 1H), 3.02 (dd, J = 14.0, 5.9 Hz, 1H). 13C NMR (126 MHz, CDCl3): δ 175.4,
155.9, 151.7, 141.5, 132.8, 130.7, 130.3, 127.9, 127.3, 125.2, 125.1, 120.2, 116.4, 115.8, 110.4, 67.3,
54.7, 47.3, 36.8. MSꢀESI m/z 482.0603 ([M + H]+, C24H21BrNO5, calc. 482.0603). [α]D 20 = −18.8 (c 1.76,
CH2Cl2). IR (neat): 3321, 1695, 1608, 1509, 1495, 1448, 1417, 1335, 1290, 1251.
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N-Fmoc-3,5-dichloro-D-tyrosine (10). An excess sulfuryl chloride (20
mL) was added to Dꢀtyrosine (1.0 g, 5.52 mmol) in AcOH (5 mL) at room
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temperature. After 24 h stirring, the reaction mixture was concentrated under a
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stream of nitrogen. Et2O/hexane mixture was added to the residue. The bisꢀ
chlorinated product was precipitated and used for the next step without further purification. The crude
product was dissolved in 50 mL dioxane/water (1:1), and NaHCO3 (1.1 g, 13.25 mmol) was added. The
solution was cooled in an ice bath and FmocꢀOSu (2.0 g, 6.06 mmol) was added in portions. The reaction
mixture was stirred overnight at room temperature. 25 mL water was added, and the solution was
extracted into EtOAc (3 x 50 mL). The combined organic layers were washed with brine (40 mL), then
dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting white solid was recrystallized
from hot Et2O/hexane (2.29 g, 88%). 1H NMR (500 MHz, CD3CN): δ 7.82 (d, J = 7.6 Hz, 2H), 7.58 (dd, J =
7.7, 2.7 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.34 – 7.28 (m, 2H), 7.20 (s, 2H), 6.03 (d, J = 8.6 Hz, 1H), 4.39
(td, J = 9.0, 4.8 Hz, 1H), 4.31 (dd, J = 10.3, 7.0 Hz, 1H), 4.25 – 4.15 (m, 2H), 3.09 (dd, J = 14.2, 4.9 Hz,
1H), 2.84 (dd, J = 14.2, 9.3 Hz, 1H). 13C NMR (126 MHz, CD3CN): δ 172.9, 156.9, 148.3, 144.93, 144.92,
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142.03, 142.00, 131.5, 130.2, 128.63, 128.62, 128.0, 126.1, 126.0, 122.1, 120.92, 120.90, 67.4, 55.6,
20
47.9, 36.5. [α]D
= −62.8 (c 0.14, CH2Cl2). MSꢀESI m/z 472.0716 ([M + H]+, C24H20Cl2NO5, calc.
472.0719). IR (neat): 3434, 3307, 3037, 2926, 1710, 1688, 1535, 1487, 1283, 1268, 1244.
Syntheses of heptapeptide substrates 11-16. 7mer peptide hydrazide substrates were
synthesized and purified as previously described.11 See Figs. S18ꢀ22.
Synthesis and purification of Coenzyme A adducts of 7mer peptides (17-21). A 4 mM solution of
7mer peptide in 0.2 M sodium phosphate acidic solution containing 6 M guanidinium chloride at pH 3 was
added to an Eppendorf tube equipped with a stir bar. The solution was cooled to ꢀ10°C in an ice/salt bath.
Sodium nitrite (25 equiv.) was added to the reaction mixture, which was stirred for 20 minutes at ꢀ10°C.
Coenzyme A trilithium salt (20 equiv.) was added to the reaction mixture to a final concentration of 100
mM. Subsequently, the pH was adjusted to 6.8ꢀ7.0 with a microꢀpH probe and the reaction was allowed to
warm to room temperature and stir for 1 h. The reaction mixture was diluted with MeCN (+ 0.1% FA) to a
final volume of 0.8 mL. Purification of peptideꢀCoA was achieved by repeated injections onto an analytical
Phenomenex Luna C18 column (5 ꢁm, 250 x 4.6 mm) that had been equilibrated with 10% MeCN in H2O
(+ 0.1% FA). The peptideꢀCoA adducts were eluted with a gradient of 10–55% MeCN in H2O (+ 0.1% FA)
over 17 minutes. The purified material was verified by HPLCꢀMS, aliquoted, and lyophilized (see Figs.
S23ꢀS27).
Expression and purification of OxyB, OxyA, PCP7-X, Fd, FdR, Sfp R4-4. OxyB, OxyA and PCP7ꢀX
from Amycolatopsis orientalis DSM 40040 were expressed and purified as previously described.11
Ferredoxin from spinach, ferredoxin reductase from E. coli, Spf R4ꢀ4 from B. subtilis (codonꢀoptimized,
K28E/T44E/C77Y triple mutant of phosphopantetheinyl transferase) were expressed and purified as
previously described.11,16b
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Activity assays of OxyB and OxyA. Assay conditions were based on previously described
protocols.11 Reactions were carried out on a 100 ꢁL scale. Loading buffer (50 mM HEPES, 20 mM KCl,
10 mM MgCl2, pH 7.0) was added to an Eppendorf tube containing 20 nmol of lyophilized peptideꢀCoA
adduct, to a final concentration of 400 ꢁM. Subsequently, final concentrations of 400 ꢁM PCP7ꢀX and 40
ꢁM of Sfp R4ꢀ4 were added to the reaction mixture, which was placed in a 30°C incubator for one hour. In
standard reactions, final concentrations of the following reagents were added to the reaction mixture, in
this order: 2 mM glucoseꢀ6ꢀphosphate, 4 units of glucoseꢀ6ꢀphosphate dehydrogenase, 14.4 ꢁM spinach
ferredoxin, 3 ꢁM E. coli flavodoxin reductase, 5 ꢁM OxyB, 7.5 ꢁM OxyA. Finally, the oxidative crosslinking
reaction was initiated by the addition of 2 mM NADPH. Typical assays were carried out at room
temperature for 2 hours in the dark. In order to remove the peptide from the carrier domain, 20,000
equivalents of propylamine were added and the reaction mixture incubated for 15 minutes. Proteins were
precipitated by adding 15 ꢁL of formic acid and 50 ꢁL of MeCN (+ 0.1% FA). Denatured proteins were
pelleted and the supernatant was analyzed by HRꢀHPLCꢀMS and MS/MS (see Figs. S28ꢀS32, Tables S1ꢀ
S6).
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Associated Content
Supporting Information. Characterization of new compounds, HPLCꢀMS analysis of new 7mer
peptides, HRꢀMS and HRꢀMS/MS data for substrates and products of enzymatic reactions. The
Supporting Information is available free of charge on the ACS Publications website at:
Author Information
Corresponding authors
Email: ejs@princeton.edu and mrseyed@princeton.edu
Notes
The authors declare no competing financial interests.
Acknowledgments
We thank the National Institutes of Health (grants GM065483 to E.J.S. and DP2ꢀAIꢀ124786 to
M.R.S.) and the Searle Scholars Program (M.R.S.) for support of this work. C.C.F. was
supported by an Eli LillyꢀEdward C. Taylor Fellowship in Chemistry.
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References
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