Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin

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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 Downloaded from http://pubs.acs.org on May 28, 2018

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The Journal of Organic Chemistry

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†,‡,*

†

Department of Chemistry, Princeton University, Princeton, NJ 08544

‡

Department of Molecular Biology, Princeton University, Princeton, NJ 08544

*Corresponding author. E-mail addresses: [email protected] (E.J. Sorensen); [email protected] (M.R. Seyedsayamdost)

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The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract We report a general method for synthesizing diverse D-Tyr analogs, one of the constituents of the antibiotic vancomycin, using a Negishi cross-coupling protocol. Several analogs were incorporated into the vancomycin substrate-peptide 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 live-saving 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 derivatives.

antibiotic 1a,2

now

have

second,

third,

and

fourth-generation

semi-synthetic

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, site-specifically 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 solid-phase 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 chemo-enzymatically into the final product. Herein, we report a method for synthesizing D-Tyr using Negishi cross-coupling reactions and demonstrate the preparation of derivatives containing a variety of substituents. Incorporation of five D-Tyr analogs at residue 6 of the vancomycin precursor peptide followed by reaction with OxyB and OxyA revealed two crosslinks, involving rings C-D and, surprisingly, rings A-B, rather than D-E (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 L-Asn, vancomycin contains six non-proteinogenic amino acids: 3,5dihydroxy-L-phenylglycine (L-Dpg), β-hydroxy-3-chloro-L-Tyr (β-OH-3-Cl-L-Tyr), 4-hydroxy-Dphenylglycine (D-Hpg), β-hydroxy-3-chloro-D-Tyr (β-OH-3-Cl-D-Tyr), and N-methyl-D-Leu.7 To prepare vancomycin derivatives, synthetic access to analogs of each amino acid is essential. For some amino acids such as L-Tyr, a plethora of derivatives are commercially available and enzymatic syntheses of variants have been reported as well.8 For others, however, notably aromatic D-amino acids, new synthetic protocols are required. Given that OxyA crosslinks 3-ClD-Tyr

at residue 6 of vancomycin, we focused our efforts on the synthesis of D-Tyr variants.

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Figure 1. Structure of vancomycin. Bonds formed by OxyB, A, and C are highlighted, along with the ring nomenclature.

D-Tyr

can be halogenated at the phenolic 3- and 5-positions 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 cross-coupling scheme between the organozinc derivative of protected 3-iodo-D-Ala (2, Fig. 2a) with aryl halides containing the desired functionalities to convergently build D-Tyr analogs.9 Using the protocol described by Jackson and coworkers, we began by cross-coupling the organozinc derivative of N-Fmoc-3-iodo-D-Ala methyl ester with readily available 4-bromo-2R phenols (R = OCH3, CH3, F, CF3) through the combined action of Pd2dba3 and P(o-tolyl)3 in DMF (Fig. 2a).10 The N-Fmoc-D-Tyr methyl esters thus obtained were then hydrolyzed using LiOH in THF/water. This method effectively afforded a variety of N-Fmoc-protected D-Tyr derivatives, ready for use in SPPS (3-6, Fig. 2b, Figs. S1-S10). Similarly, N-Fmoc-3,4dihydroxy-D-Phe (N-Fmoc-D-DOPA) was synthesized by Negishi coupling of 2 with 4-bromo-1,2dimethoxybenzene, followed by demethylation (7, Fig. 2b, Figs. S11-13). This approach also proved convenient for generating protected 3-chloro-4-amino-D-Phe (8, Fig. 2b, Figs. S14-S15). On the other hand, N-Fmoc-3-Br-D-Tyr and N-Fmoc-3,5-Cl2-D-Tyr were synthesized by EAS using traditional methods (9, 10, Fig. 2c, Figs. S16-S17). The combination of these procedures enabled the creation of a small library of diverse D-Tyr amino acids (Fig. 2). With the D-Tyr analogs in hand, we next established their use in SPPS, in which the Nprotected amino acids were coupled onto a hydrazinated 2-chlorotrityl chloride resin using Fmoc-chemistry (Fig. 3a). Of the D-Tyr analogs shown (Figs. 2b, 2c), we focused on those with ortho substituents to examine their reactivity with OxyB and OxyA. Amino acids 3-5, 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) D-Tyr derivatives synthesized using Pd-catalyzed cross-coupling. (c) Synthesis of halogenated D-Tyr residues by electrophilic aromatic substitution. Fmoc = fluorenylmethyloxycarbonyl; dba = dibenzylideneacetone; DMF = N,N-dimethylformamide; FmocOSu = 9-fluorenylmethyl succinimidyl carbonate.

were incorporated into simplified 7mer peptides. As controls, we also inserted D-Tyr and 3-Cl-DTyr at residue 6, both of which we employed previously to recapitulate the reaction of OxyA in vitro.11 Aside from the D-Tyr analogs at residue 6, two additional changes were made relative to the native vancomycin 7mer peptide: (1) we used 3-Cl-L-Tyr at residue 2, rather than β-OH-3Cl-L-Tyr, as the latter repeatedly resulted in dehydration of the β-OH moiety; and (2) we chose L-Tyr

at residue 1, rather than L-Dpg, which has been reported to result in racemization during

SPPS. Each 7mer peptide was released as an acyl-hydrazide (11-16, Fig. 3b, Figs. S18-S22) and subsequently converted into a coenzyme A (CoA) conjugate using a native chemical ligation-based strategy to deliver substrates 17-23 in good yields (Fig. 3c, Figs. S23-27).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 D-Tyr building blocks. Upon purification, the 7mer-CoA adducts were used to attach the peptide substrate to an X-domain/peptidyl-carrier protein (X-PCP) di-domain. In this construct, the PCP domain carries the phosphopantetheine-7mer on a conserved active site Ser residue, while the X-domain

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recruits the cytochrome P450 enzymes to facilitate efficient cross-coupling.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 X-PCP via propylamine-mediated aminolysis, because amidation of the C-terminus has been shown to enhance the reactivity of vancomycin.5a,15 The products of the reaction were subsequently assessed by HPLC-Qtof-MS.

Figure 3. Incorporation of D-Tyr analogs into vancomycin substrate peptides. (a) SPPS of vancomycin analogs containing D-Tyr variants to give acyl hydrazides. (b) 7mer peptide hydrazides described in this work. (c) Scheme for synthesis of CoA-adducts of 7mer peptides. DIPEA = N,N-diisopropylethylamine; COMU = (1-cyano-2-ethoxy-2oxoethylidenaminooxy)dimethylamino-morpholino-carbenium

hexafluorophosphate;

DBU

=

1,8-

diazabicyclo[5.4.0]undec-7-ene; TIS = triisopropylsilane.

Analysis of the reaction with D-Tyr at residue 6 (23) with OxyB and OxyA resulted only in the C-O-D aryl ether bond, as previously established.11 Conversion to the singly-crosslinked product proceeded in 86% yield. On the other hand, peptide 22 containing 3-Cl-D-Tyr gave both C-O-D and D-O-E crosslinks, as confirmed by HR-MSn analysis. The doubly-crosslinked 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 17-21 (Fig. 4a). Incubation with OxyB and OxyA gave both singly and doubly-crosslinked products as inferred from a loss of 2 Da or 4 Da relative to each substrate, which was determined by highresolution MS (HR-MS, 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 HR-tandem-MS (HR-MS/MS) to assess the site of crosslink formation. For all substrates containing D-Tyr 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 singly-crosslinked 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 C-O-D aryl ether bond by OxyB (24, Fig. 4b, Figs. S28-33, Tables S1-S6). Examination of the doubly-crosslinked products also revealed the C-O-D 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 singly-cyclized product. These results point to the presence of two crosslinks involving the first four residues of the peptides, one consisting of a C-O-D link and the other involving rings A and B (Fig. 4c, Figs. S28-33, Tables S1-S6). 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 A-B crosslink in peptides 17-21 would also be a bis-aryl ether linkage and could consist either of the Hpg phenol-oxygen bound to the meta position of the Tyr side chain (25, B-O-A, Fig. 4c), or the Tyr-phenol-oxygen linked to the meta position of Hpg (26, A-O-B, Fig. 4C). We utilized an isotope replacement approach using 3,5-2H2-L-Tyr to distinguish between these possibilities (Fig. 4d). Reaction of OxyB with peptide 20, now bearing 3,5-2H2-L-Tyr at residue 1 and 3-Br-DTyr at residue 6, followed by analysis by HR-MSn 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 A-O-B, rather than B-O-A, macrocycle (Table S1, Fig. 4d). By extension, we propose that products 27-31 also contain C-O-D and A-O-B macrocycles (Fig. 4d). Finally, we quantified product yields with each of the D-Tyr variants using HR-MS (Table 1). We note that each crosslink reduces the number of ionizable heteroatoms, thereby making electrospray ionization progressively less efficient with C-O-D and A-O-B-bearing modifications.

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Figure 4. Formation of two crosslinks catalyzed by OxyB. (a) Attachment of CoA-adducts of 7mer peptides substrates to the PCP-X di-domain 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 HR-MS/MS. (c) Two possible structures of doubly-crosslinked peptide products formed by OxyB, with HR-MS/MS fragments 2

shown. (d) Incorporation of 3,5- H2-Tyr as a C-terminal residue in a 7mer peptide carrying 3-Br-D-Tyr at residue 6. Results of enzymatic assays reveal formation of an A-O-B crosslink in this variant and, by extension, in other residue 6 derivatives.

As such, MS quantification tends to underestimate the amounts of singly- and doublycrosslinked products relative to substrate. With this caveat in mind, certain trends can nonetheless be inferred from the data. Substitutions at the ortho position of D-Tyr lowers the yields of C-O-D 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 OxyB-catalyzed C-O-D yields. On the other hand, the yield of the doubly-crosslinked product containing the A-O-B bond did not show a clear correlation with either electronic or steric properties for D-Tyr at residue 6. The results above have important implications for the generation of vancomycin derivatives using our proposed chemo-enzymatic 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 C-O-D and A-O-B macrocycles, with

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the latter marking a new motif within the vancomycin framework. Thus new, doubly-cyclized derivatives can be accessed with OxyB alone by altering the amino acids that occupy positions 6 and 1. The properties of the doubly-cyclized 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 3-ClD-Tyr

at residue 6.

Table 1. Product yields obtained upon reaction of vancomycin substrate peptides with OxyB.a

a

D-Tyr Analog

% C-O-D conversion

% A-O-B conversion

van der Waals Radius (Å)

3-H 3-F

86% 60%

0 3%

1.2 1.47

3-OMe

81%

2%

1.52

3-Cl

17%

0

1.75

3,5-Cl2

11%

4%

1.75

3-Br 3-Me

16% 23%

4% 10%

1.85 2.0

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., Chem-Impex International, Fisher Scientific, Oakwood Chemicals and Sigma Aldrich, and were used directly without further purifications. Fmoc- and side chain-protected amino acids, 2-chlorotrityl chloride resin and other components for solid-phase peptide synthesis were purchased from Novabiochem/EMD Millipore and Sigma-Aldrich. Reactions were monitored by thin layer chromatography (TLC) carried out on 250 µm 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 µm particle size, 230 – 400 mesh). 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, βmercaptoethanol, Sephadex G-25, COMU, NEt3, DIPEA, TIS, DBU, TFA, Fmoc-OSu, hydrazine monohydrate, NaNO2, coenzyme A trilithium salt, glucose-6-phosphate dehydrogenase, glucose-6+

phosphate, ferredoxin from spinach, ferredoxin-NADP reductase from spinach, and other components necessary for biochemical assays were obtained from Sigma-Aldrich. 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). 1

H 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 1

at δ 1.94 ppm. Data for H 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.

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C 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

13

C NMR spectra are reported in terms of chemical shift and multiplicity where

appropriate. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer with a 30000-200 cm

-1

-1

diamond and are reported in terms of frequency of absorption (cm ). High-resolution 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.

HO

CO2Me

NHFmoc

N-Fmoc-D-serine methyl ester. D-Serine methyl ester hydrochloride (5.35 g, 34.4 mmol) was dissolved in 100 mL water/dioxane (3:1) and NaHCO3 (7.22 g, 86.0 mmol) was added. The solution was cooled to 0 °C and Fmoc-OSu (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 1

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).

13

C 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. MS-ESI m/z 364.1159 ([M + Na] , C19H19NNaO5, calc. 364.1161). [α]D

20

= −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 100-mL 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 recooled to 0 °C. A solution of N-Fmoc-D-serine 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 1

yield the product as white solid (3.84 g, 85%). H 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).

13

C 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. MS-ESI 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 flame-dried, nitrogen-purged side arm round-bottomed 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. N-Fmoc-3-iodo-D-alanine 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(o-tolyl)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. N-Fmoc-3-R-D-Tyr 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 4-6 hours. The pH was then adjusted to 2-3 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 N-Fmoc-R-D-Tyr. Me HO

N-Fmoc-3-methyl-D-tyrosine methyl ester. The compound was synthesized

CO2Me

from 4-Bromo-2-methylphenol using the standard cross-coupling procedure and

NHFmoc

purified by flash column chromatography using hexanes/EtOAc (4:1) as the 1

eluting solvent. Pure material was obtained as a white solid (310 mg, 72%). H 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).

13

C 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, +

67.2, 55.1, 52.5, 47.3, 37.6, 15.9. MS-ESI m/z 432.1807 ([M + H] , C26H26NO5, calc. 432.1811). [α]D

20

=

−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 1

mg, 63%). H 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).

13

C 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). MS-ESI m/z

+

418.1652 ([M + H] , C25H24NO5, calc. 418.1654). IR (neat): 3327, 2925, 1697, 1611, 1510, 1449, 1262, 1212. MeO HO

CO2Me NHFmoc

N-Fmoc-3-methoxy-D-tyrosine methyl ester. The compound was synthesized from 4-Bromo-2-methoxyphenol using the standard cross-coupling procedure 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).

13

C 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,

12


Page 13 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


+

55.0, 52.5, 47.3, 38.1. MS-ESI 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. 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 1

mg, 65%). H NMR (500 MHz, CD3CN): δ 7.82 (d, J = 7.6 Hz, 2H), 7.59 (d, J = 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).

13

C 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). MS-ESI m/z

+

434.1604 ([M + H] , C25H24NO6, calc. 434.1604). IR (neat): 3325, 2926, 1700, 1514, 1449, 1430, 1270, 1234, 1208. F HO

CO2Me NHFmoc

N-Fmoc-3-fluoro-D-tyrosine methyl ester. The compound was synthesized from 4-Bromo-2-fluorophenol using the standard cross-coupling procedure and purified by flash column chromatography using hexane/EtOAc (4:1) as the 1

eluting solvent. Pure material was isolated as a white solid (195 mg, 45%). H 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).

13

C 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, 19

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. F NMR (282 MHz, +

CDCl3): δ -140.25 (t, J = 10.3 Hz). MS-ESI m/z 436.1563 ([M + H] , C25H23FNO5, calc. 436.1560). [α]D

20

=

−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).

13

C 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.

19

F NMR (282 MHz, CD3CN): δ -138.77 (d, J = 12.0 Hz). [α]D +

20

= −26.0 (c

0.25, CH2Cl2). MS-ESI m/z 422.1400 ([M + H] , C24H21FNO5, calc. 422.1404). IR (neat): 3397, 3324, 3306, 2926, 2851, 1698, 1516, 1448, 1259.

13



F3 C

CO2Me NHFmoc

HO

N-Fmoc-3-trifluoromethyl-D-tyrosine

Page 14 of 20

methyl

ester.

The

compound

was

synthesized from 4-Bromo-2-(trifluoromethyl)phenol using the standard crosscoupling procedure and purified by flash column chromatography using 1

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). 13

C 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.

19

F NMR (282 MHz, CDCl3): δ -60.96. [α]D

20

= −28.3 (c 0.20, CH2Cl2).

+

MS-ESI m/z 486.1532 ([M + H] , C26H23F3NO5, calc. 486.1528). IR (neat): 3351, 2955, 2937, 2861, 1693, 1622, 1514, 1444, 1323, 1305, 1271. 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 1

mg, 72%). H 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).

13

C 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. 19

+

F NMR (282 MHz, CD3CN): δ -62.65. MS-ESI 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. MeO MeO

CO2Me NHFmoc

N-Fmoc-3,4-dimethoxy-D-phenylalanine methyl ester. This compound was synthesized from 4-bromoveratrole using the standard cross-coupling procedure. The crude reaction mixture was applied directly to a silica gel

column, purified using hexane/EtOAc (5:1) as the eluting solvent. Pure material was isolated as a white 1

solid (336 mg, 73%). H 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).

13

C 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. MS-ESI m/z 462.1913 ([M + H] , C27H28NO6, calc. 462.1917). [α]D (c 0.42, CH2Cl2). IR (neat): 3322, 2926, 1752, 1693, 1535, 1517, 1447, 1267, 1236.

14


20

= −28.5

Page 15 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-Fmoc-D-DOPA (7). Dry dichloromethane (5 mL) and N-Fmoc-3,4-dimethoxyD-phenylalanine methyl ester (336 mg, 0.73 mmol) were added to a 25-mL flame-dried flask under inert atmosphere. The reaction mixture was cooled to 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 drop-wise. 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 N-Fmoc-D-DOPA methyl ester (101 mg, 32%). Then eluting solvent was changed to hexane/EtOAc/AcOH (2:2:0.05) to obtain N-Fmoc-D-DOPA (156 mg, 51%). Using the standard hydrolysis procedure, N-Fmoc-D-DOPA 1

methyl ester was converted into N-Fmoc-D-DOPA as well. N-Fmoc-D-DOPA methyl ester: H 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).

13

C 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. MS-ESI +

m/z 434.1602 ([M + H] , C25H24NO6, calc. 434.1604). [α]D

20

= −23.4 (c 0.80, CH2Cl2). IR (neat): 3316, 1

3052, 2951, 1737, 1726, 1691, 1530, 1447, 1343, 1269, 1219. N-Fmoc-D-DOPA: 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).

13

C 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. MS-ESI 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. Cl H2N

CO2Me NHFmoc

N-Fmoc-3-chloro-4-amino-D-phenylalanine methyl ester. This compound was synthesized from 2-chloro-4-bromoaniline using the standard cross-coupling procedure. The crude reaction mixture was applied directly to a silica gel 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).

13

C 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

15


20

= −18.1 (c 0.26, CH2Cl2). MS-


Page 16 of 20

+

ESI m/z 451.1425 ([M + H] , C25H24ClN2O4, calc. 451.1425). IR (neat): 3359, 2950, 1709, 1623, 1506, 1447, 1308, 1250, 1210. N-Fmoc-3-chloro-4-(tert-butoxycarbonyl)amino-D-phenylalanine

(8).

The

purified cross-coupled N-Fmoc-3-chloro-4-amino-D-phenylalanine methyl ester product was hydrolyzed using the standard hydrolysis procedure. After 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). 1

H 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).

13

C 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. MS-ESI m/z 537.1798 ([M + H] , C29H30ClN2O6, calc. 537.1792). [αD

20

= −4.9 (c 2.14, CH3OH). IR (neat): 3420, 2919, 2849, 1711, 1520,

1449, 1227. N-Fmoc-3-bromo-D-tyrosine (9). D-Tyrosine (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 Fmoc-OSu (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).

13

C 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. MS-ESI m/z 482.0603 ([M + H] , C24H21BrNO5, calc. 482.0603). [α]D CH2Cl2). IR (neat): 3321, 1695, 1608, 1509, 1495, 1448, 1417, 1335, 1290, 1251.

16


20

= −18.8 (c 1.76,

Page 17 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


N-Fmoc-3,5-dichloro-D-tyrosine (10). An excess sulfuryl chloride (20 mL) was added to D-tyrosine (1.0 g, 5.52 mmol) in AcOH (5 mL) at room temperature. After 24 h stirring, the reaction mixture was concentrated under a stream of nitrogen. Et2O/hexane mixture was added to the residue. The bischlorinated 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 Fmoc-OSu (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 1

from hot Et2O/hexane (2.29 g, 88%). H 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).

13

C NMR (126 MHz, CD3CN): δ 172.9, 156.9, 148.3, 144.93, 144.92,

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, 47.9, 36.5. [α]D

20

= −62.8 (c 0.14, CH2Cl2).

+

MS-ESI 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. S18-22.

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 6.8-7.0 with a micro-pH 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 peptide-CoA was achieved by repeated injections onto an analytical Phenomenex Luna C18 column (5 µm, 250 x 4.6 mm) that had been equilibrated with 10% MeCN in H2O (+ 0.1% FA). The peptide-CoA adducts were eluted with a gradient of 10–55% MeCN in H2O (+ 0.1% FA) over 17 minutes. The purified material was verified by HPLC-MS, aliquoted, and lyophilized (see Figs. S23-S27). Expression and purification of OxyB, OxyA, PCP7-X, Fd, FdR, Sfp R4-4. OxyB, OxyA and PCP7-X from Amycolatopsis orientalis DSM 40040 were expressed and purified as previously described.

11

Ferredoxin from spinach, ferredoxin reductase from E. coli, Spf R4-4 from B. subtilis (codon-optimized, K28E/T44E/C77Y triple mutant of phosphopantetheinyl transferase) were expressed and purified as previously described.

11,16b

17



Activity assays of OxyB and OxyA. Assay conditions were based on previously described protocols.

11

Reactions were carried out on a 100 µL 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 peptide-CoA adduct, to a final concentration of 400 µM. Subsequently, final concentrations of 400 µM PCP7-X and 40 µM of Sfp R4-4 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 glucose-6-phosphate, 4 units of glucose-6-phosphate dehydrogenase, 14.4 µM spinach ferredoxin, 3 µM E. coli flavodoxin reductase, 5 µM OxyB, 7.5 µM 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 µL of formic acid and 50 µL of MeCN (+ 0.1% FA). Denatured proteins were pelleted and the supernatant was analyzed by HR-HPLC-MS and MS/MS (see Figs. S28-S32, Tables S1S6).

Associated Content Supporting Information. Characterization of new compounds, HPLC-MS analysis of new 7mer peptides, HR-MS and HR-MS/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: [email protected] and [email protected] Notes The authors declare no competing financial interests. Acknowledgments We thank the National Institutes of Health (grants GM065483 to E.J.S. and DP2-AI-124786 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 Lilly-Edward C. Taylor Fellowship in Chemistry.

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Modulating OxyB-Catalyzed Cross-Coupling Reactions in Vancomycin

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