Heck Diversification of Indole‐Based Substrates under Aqueous Conditions: From Indoles to Unprotected Halo‐tryptophans and Halo‐tryptophans in Natural Product Derivatives

Abstract The blending of synthetic chemistry with biosynthetic processes provides a powerful approach to synthesis. Biosynthetic halogenation and synthetic cross‐coupling have great potential to be used together, for small molecule generation, access to natural product analogues and as a tool for chemical biology. However, to enable enhanced generality of this approach, further synthetic tools are needed. Though considerable research has been invested in the diversification of phenylalanine and tyrosine, functionalisation of tryptophans thorough cross‐coupling has been largely neglected. Tryptophan is a key residue in many biologically active natural products and peptides; in proteins it is key to fluorescence and dominates protein folding. To this end, we have explored the Heck cross‐coupling of halo‐indoles and halo‐tryptophans in water, showing broad reaction scope. We have demonstrated the ability to use this methodology in the functionalisation of a brominated antibiotic (bromo‐pacidamycin), as well as a marine sponge metabolite, barettin.


Introduction
Tryptophan is ak ey residue in many biologically active natural products,p eptides and proteins. [1,2] Its intrinsic fluorescence dominates the spectrophotometric properties of ag iven peptide or protein;i ti sacrucial residue for stabilising secondary and tertiary structure through intra andi ntermolecular interactions. [3] Tryptophan residueshave been shown to play ac entral role in protein folding [4] as well as being implicated in govern-ing the function of many biologically important systems including mechanosensitivec hannels within the membrane. [5] The possibility of functionalising tryptophan in order to modify this importantr esidue both sterically and electronically would be exciting and potentially afford am eans of interrogating, modulating andt uning the properties of peptides, proteins and natural products.
Though considerable research has been carried out on selective modification of halogenated phenylalanines and tyrosines [7] through the application of cross-coupling chemistry, the functionalisation of tryptophan has, until recently,r emained largely unexplored. [6] One potential reason for this is the challenget hat such metal mediated cross-coupling reactions presentw ith tryptophan;i ndeed, tryptophan has been demonstratedt op oisont he Suzuki-Miyaura cross-couplingo f halo-indoles. [8] This may be attributedt ot he amino acid coordinating to the palladium catalyst. [9] Notably,e sterification of the carboxylate and acylation of the primary aminer educes reactionp oisoning,h owever even this speciesi sn ot fully innocent and its incorporation in ar eactioni ss till seen to impact upon conversion. [8] In recent years, series of studies enabling the cross-coupling of halo-tryptophans througha pplication of Suzuki-Miyaura and Sonogashira chemistries in aqueous media have been reported. [6][7][8] Combinationo ft hese chemistries with enzymatic halogenation has been used powerfully for selectiveC ÀHa ctivationa nd diversification of small molecules [10] The application of Heck cross-coupling would provide av aluable addition to this growing portfolioo fr eactions for tryptophanf unctionalisation,e nablingt he potentialf or extension of conjugationa nd tuning of electronic and fluorescencep roperties as well as the opportunity to potentially modulate conformations of small molecules, peptides and proteins.
Heck cross-coupling methodologies have been effectively appliedt os eries of other biomolecules including nucleosides, [11] nucleotides and nucleosidet riphosphates. [12] However, there are very few studies reported of the utilisation of Heck methodologies for the modification of amino acids, and peptides, mostly utilising highly activated (iodo or triflate) substrates. These include modificationo fN -a nd C-protected 3,5-di-iodo-l-tyrosine, modulating ande xtending its conjugation and enabling its fluorescencep roperties to be tuned, and diversification of N-and C-protected l-tyrosine para-triflate, [13] and selective modificationo f4 -iodo-l-phenylalanine within a small protein, using Mizoroki-Heck conditions. [14] Whilst this manuscript wasb eing prepared, the first Mizoroki-Heck coupling of ah alo-tryptophan was reported. In this study free, unprotected7 -bromo-tryptophan was derivatised with 6d ifferent styrenes. [15] Excitingly,a so bserved with the Suzuki-Miyaura cross-coupling of tryptophan, [6a, 8] these tryptophan-7-styrene products were shown to be fluorogenic, thus again opening up the way for fluorescencem odulation of halo-tryptophans. There is considerable potential for the development of Heck methodologies for the functionalisation of free and biomoleculee mbedded halo-tryptophans. Herein, we report our exploration of the systematic application of the Heck reaction first to halo-indoles,t hen to free and unprotected halo-tryptophans. We then move on to challenge the developed methodology,a pplying it to unprotected and complex natural product barettin as well as natural product derivative bromo-pacidamycin and to ar ange of aliphatic and aromatic alkenes (Scheme 1).
To improvet hese results,w en ext explored the impact of varying the catalyst, (investigating water-soluble Na 2 PdCl 4 )i n combination with exploring the application of the sterically more demanding and electron-rich ligand S SPhos, designedb y Buchwald. [17] Microwaveheating was also explored ( Table 2). These studies revealed that by using S SPhos in place of TXPTS gave am odest increase in conversion of 5-iodo-indole 1 (78-85 %, Ta ble2,e ntries 1a nd 2). As ignificant increasei n conversion (97 %) could be seen upon replacing Pd(OAc) 2 with Na 2 PdCl 4 ( Table 2, entry 3). By replacing conventional heating with microwaveh eating, > 99 %c onversionc ould be achieved after only one hour.N otably,f or 5-iodo-indole 1,i tw as possible to achieve almost quantitative conversion using microwave heatinga nd in the absence of an additional ligand ( Table 2, entry 5). Next, we set out to explore the Heck modification of the less reactive 5-bromo-indole 4.H ere, we observed conversions to be far more modest in the absence of any ligand, however utilisationo f S SPhos again enableda lmostq uantitative conversions ( Table 2, entry 8).
With these conditions in hand, we next set out to explore whether we could proceed past acrylic acid 2 as the coupling partner.T he conditions that we had developed showed good applicability enabling the Heck modification of both 5-iodo 1 and 5-bromo-indole 4 to proceed almost quantitativelyw itha range of different alkene cross-couplingp artners(Ta ble 3).
The only exception to this rule wass tyrene 11 (Table 3, entry 6) because of the reduced reactivity of this species arising due to the electron rich nature of the alkene. Also, for this reason,n onactivated 1-octene and cyclohexeneg ave no conversion under the same reactionc onditions. From NMR characterisation of the products it may be seen that all Heck coupling reactions progressed with ah igh level of stereoselectivity to generateo nly the E-isomers. The trans-relation of the double bonds was established on the basis of the coupling constant for the vinylic protons in the 1 HNMR spectra (J = 16 Hz, see Supporting Information).
Havingachieved asystem that would work well for the functionalisation of iodo-and bromo-indoles 1 and 4,w en ext set out to explore whether it might be possible to extendt his methodology further to the aqueous cross-coupling of the far more challenging free, unprotectedh alo-tryptophans.
Halo-tryptophans mayb er eadily accessed through as imple one-step biotransformation using tryptophan synthase, [18,19] or through a4 -5 step chemical synthesis. [20] Challengest hat need to be addressedt or ender Heck cross-coupling of free halotryptophans usefula re their poor solubility and their propensity to chelate to and deactivate the palladiumc atalyst. [9] Utilisation of our previousc onditions that had been optimised for the aqueous Heck cross-couplingo fh alo-indoles with acrylic acid resulted in almostn oo bservable conversion of 5-bromo-tryptophan 19 even at 100 8C( Ta ble 4, entries 1 and 2), we therefore returned to exploration of both TXPTS and TPPTS as water soluble ligandsw ith the highly reactive 7-iodo-tryptophan 20.B yd oubling both the amount of catalyst and ligand, it was possible to achieve almost quantitative conversion using either TPPTS or TXPTS when heated to 90 8C, thoughw ith TPPTS an extended reactiont imeo f2hw as required( Ta ble 4, entries3-6). Applying these conditions, using TPPTS to the less reactive 7-bromo-tryptophan 21,ac onversion of only 47 %i so bserved (Table 4, entry 7). However,b y switchingt ot he more sterically demanding TXPTS ligand, almostq uantitativec onversion was achieved (Table 4, entry 8). As the 5a nd 6-halo-tryptophans are more reactive than the 4 and 7-counterparts, the sterically less demanding TPPTS was  found suitable to afford almost quantitative conversion of 5-bromo and 6-bromo-tryptophan 19 and 22 (Table 4, entries 9 and 10). Importantly,a ss een for the halo-indoles the reaction is highly stereoselective towards the E product (See Supporting Information).

Observed limitations in reactivity
The 4-bromo-tryptophan 23 is highly unreactivea nd even using TXPTS as ligand, only as mall trace of product may be perceived from the reaction. Curiously,t he sole application of Heck cross-coupling to tryptophan reported in the literature is the functionalisation of 4-bromo-tryptophan 23 in the synthesis of clavicipitic acid (reported as 91 %c onversion using conventional heating); [15] we have been unable to reproduce this conversion using either the system that we have developed or their conditions with alkene 1,1-dimethylallyl alcohol, TPPTS, Pd(OAc) 2 and NaOH as base. Forcing the reactionb yM Wh eating to 130 8Cf or 2h we were finally able to obtain ac onversion of around 15 %. The less reactive aryl chloride, as ac omponent of either the simple indole system or within tryptophan is also recalcitrant to Heck modification under all conditions that we have explored.H owever,w ed emonstrate that followingp rotection of the primary amine (using N-Boc-4-bromo-tryptophan), Heck coupling at position4can be achieved using our conditions. A similar trend was observed for reactivity of free versusp rotected 2-bromoo r4 -bromo-phenylalanine. These observations strongly indicate influence of free a-amino group on the Pd-catalysed cross-coupling of 4-halo-tryptophans( see Supporting Information).
With conditions established that would enablet he conversion of 5, 6a nd 7-iodo and bromo-tryptophans, we next set out to explore the impacto fs terics and electronic and solubility of the cross-coupling partners (Scheme 2). Through this we could observe that progression from acrylic acid to bulkiera romatic substrates could be successfully achieved. Trend in coupling efficiencys eems to follow electronics rather than substrate solubility.I ti se vident from the results that electron donating substituents (i.e. Me or amino) decrease the reactivity of alkene resulting in lower yields (51-65 %, compounds 31-34). On the contrary,e lectron-withdrawing fluoro or nitrile derivatives gave significantly higher yields (80-92 %, compounds 35-39), and reactions also workedw ell with heterocyclic alkenes ubstrates such as 4-vinylpyridine (compounds 40 and 41).
From NMR characterisation of the products, it may be seen that for the 5-bromoa nd 5-iodo-tryptophan, an almoste qual mixture of the E and Z isomer are generated, whereas for the 7-bromo and 7-iodo-tryptophans the E stereoisomers predominate (see Supporting Information).
The selective diversification of natural products is an important area. Such work can enables ystematic modification and optimisation of ab ioactive molecule'sp roperties, or enable tagging and tracking, or be utilised in target identity.A sp roof of principle, we next set out to explore whether Heck crosscoupling might be applied to the cross-coupling of tryptophan residues within two test-bed naturalp roducts.T he sponge halo-metabolite barettin, ab rominated ad iketopiperazine-type cyclic dipeptide (42,S cheme 3), is knownf or its roles in chemical defence against predators, antifouling activity and binding to serotonergic5 -HT receptors. [21] Heck modulation could be potentially exploredt oe nablea nalogue generation to gain greater understandingo ft he molecule's structure-activity relationship. Furthermore, Heck tagging impactsu pon the fluorescence of halo-indoles/tryptophans, such as trategy could potentially be applied to enable the tissues in which it is generat- Within barettin, the primary amine and carboxylate of tryptophan, that would usually add challenge to cross-coupling reaction progression,a re maskeda sa mides within ad iketopiperazine. Many marine metabolites are highly lipophilic, limiting their diffusion from their producer,e nabling the producer to retain the metabolites.T od issolve barettin, ah igherr atio of acetonitrile was required. Once dissolved, the reactionp roceeded well, affordingproduct 43.
Pacidamycin,b elonging to the class of uridyl peptide antibiotics, represents ap otentially more challenging substrate, comprised of ap seudo-peptide backbone attached via an exocyclic enamide to am odifiedu ridine. We hadp reviously demonstrated the first out of context use of ah alogenase, by introducing the gene encodingt ryptophan-7-halogenase prnA in Streptomycesc oeruleorubidis (RG-5059) in order to generate Cl-pacidamycin. [6a] We successfully utilized as ynthetic biological approach to generate an engineered strain (S. coelicolor M1154,n amed RG1104) capable of generating new bromometabolites, Br-pacidamycinD (44)a nd performing Suzuki-Miyaura derivatisation to gain access to the analoguea ryl-paci-damycinD(Scheme 4). [8] In this study,w ee mployedt he same strain in ISP2 medium for production and isolation of Br-pacidamycin Da nd cultures were grown for at otal of 7-8 days. Production of Br-pacidamycin Da long with wild typep acidamycin Dw as detected by LC-HRMS analysis. Isolation of the target compounds followed sequence of purification steps:s olid-phase extraction (XAD-16 resin), enrichment of pacidamycins using ion exchange chromatography (HiTrap SP-FFc olumns) and reverse phasep urification (semi-preparative HPLC) (see Supporting Information for details).
In this manner,reasonably pure samples of wild-type pacidamycin D( 1mg) and Br-pacidamycin D( ca. 0.5 mg) were isolated. With the isolated materiali nh and, we could perform partial NMR analysiso nt heses amples. While the 1 HNMR on paci-damycinDwas satisfactory,p eak broadening was observedf or the brominated analogue. Gratifyingly,s ome key differences were noted by careful comparison of the HSQC-NMR( see Sup-porting Information), notably 1 Ha nd 13 Cp eak at 7.35 and 110.7 ppm for pacidamycin Dw as absent in the HSQC spectrum of Br-pacidamycin D, thus indicating substitution in desired position. LC-HRMS 2 analyses showed desired isotope pattern (m/z 790, 792 for 79 Br, 81 Br,r espectively)a nd satisfactory MS 2 fragmentation was obtained (see SupportingI nformation).
To enablet he modification of bromo-pacidamycin through application of Heck chemistry we first explored reactions on crude extracts containing very low concentrations of this bromometabolite. Using our optimum conditions developed for Br-tryptophan, we were pleased to see full conversion of Br-pacidamycin Df rom crude extract and LC-HRMS 2 analyses confirmed formationo ft he desired cross-coupling product. Next,w ec arried out the Heck coupling on ap urified sample of Br-pacidamycin D( ca. 0.5 mg). Although the cross-coupling was successful as confirmed by LC-HRMS 2 analysis (LC-HRMS 2 product m/z C 40 H 47 FN 9 O 10 + [M+ +H] + :8 32.3424; found: 832.3422), the corresponding product 45 could not be successfully isolated on this very small scale. These results indicated that our methodh as the potential to be utilised for microscale tagging or functionalisation of brominated metabolites, even as components of ac omplex extract without the need for prior application of purificationo rp rotection strategies.

Conclusions
We have developed conditions enabling Heck cross-coupling of iodo-and bromo-indoles and free unprotected5 ,6and 7bromo-tryptophan and 7-iodo-tryptophan,s howing the reac- tion to be very high yielding. Near quantitative cross-coupling of iodo-and bromo-indolesc an be achieved in the absence of ligand,h owever to enablec omparable conversionso fh alotryptophans, aw ater-soluble ligand is required, and we found that TXPTS and TPPTS could be used effectively.W hile high conversionsc ould be achievedf or 5, 6, and 7-iodo and bromoindoles and tryptophans, exploration of limitation of scope revealed the lessr eactive aryl chlorides to be recalcitrant to cross-coupling under our conditions, 4-halo speciesa lso proveddemanding.
We have demonstrated the ability to carry out Heck modification of halo-tryptophan with aw ide range of alkenyl crosscoupling partners. Introducing this chemistry to more sensitive and complex systems in which halo-tryptophans are embedded, we have used synthetic biology to engineer the production of the halo-metabolite, bromo-pacidamycin, and have demonstrated the selectivem odification of this polar antibiotic, even as ac omponent of ac omplex extract. We have also demonstrated the application of these conditions to the modification of barettin, an atural and lipophilic metabolite obtained from as ponge.
The fairly mild aqueousc onditions, the high conversions and flexibility of the substrate scope, make the Heck reactiona useful tool for applicationt oc hemicalb iology and molecule tagging as well as to GenoChemetic approaches to natural product analogue generation.T ryptophan is an important residue in natural products, peptidesa nd proteinsw ith ak ey role in folding, functiona nd fluorescence. The extension and modulation of the conjugation enabledb yH eck cross-coupling with series of alkene partners provides the potentialf or tuning the conformation and modulating fluorescenceproperties.
High-and low-resolution mass spectra that were recorded at the University of St. Andrews on an Orbitrap VELOS pro. Freeze drying was carried out on aS canvac CoolSafe freeze dryer.M icrowave reactions were performed in sealed vials using aB iotage Initiator + microwave reactor.U PLC analysis was acquired on aW aters Acquity H-Class UPLC system fitted with aW aters Acquity UPLC BEH C18 column (1.7 mm, 2.1 50 mm) or Phenomenex Kinetex Phenyl-hexyl column (2.1 mm, 2.1 75 mm).
Flash chromatography was performed using Davisil silica gel LC60A (40-63 micron). Thin layer chromatography (TLC) was executed using aluminium sheets of silica gel 60 F254 and was visualised under aMineralight model UVGL-58 lamp (254 nm). The plates were developed with ninhydrin in acetone or basic potassium permanganate solutions. Purification of unprotected tryptophan derivatives and peptides was carried out on aB iotage Isolera Four using reverse-phase SNAP C18 12 gc olumn cartridges. The purifi-cation was carried out using water (solvent A) and methanol/acetonitrile (solvent B) using the following gradient:0 -1.5 min (5 %B ), 1.5-3.0 min (5 %t o1 5% B), 3 Preparative RP-HPLC purification was performed using aG ilson 322 pump, 151 UV/Vis detector and 233XL fraction collector,u sing a Phenomenex Luna C18 (5 micron, 250 21.2 mm) with UV detection at 234 nm. Elution was carried out using as hallow linear gradient with starting conditions 95 %s olvent A( 0.1 %f ormic acid in MQ water) to 5% solvent B( ACN) to 40 %s olvent Bo ver 40 min.
Experimental details and characterisation data N-Boc protection of 4-Br-(S)-tryptophan, it's subsequent Heck coupling and deprotection affording compound 28,a sw ell as Heck reaction for N-Boc-4-Br-phenylalanine and N-Boc-2-Br-phenylalanine are presented in the Supporting Information.
General protocol for Heck cross-coupling of halo-indoles with alkenes in aqueous conditions:I na10 mL pear-shape flask or 0.5-2mLM Wv ial, sodium tetrachloropalladate (1.6 mg, 5 mmol, 5mol %), sulfonated SPhos (6.6 mg, 12.5 mmol, 12.5 mol %) were purged with nitrogen and stirred at RT for 15 min after adding 1mLo fd egassed water/acetonitrile (1:1) mixture. Then, appropriate halo-indole (0.1 mmol, 1.0 equiv) is added together with Na 2 CO 3 (22 mg, 0.2 mmol, 2equiv) followed by addition of the alkene (0.15 mmol, 1.5 equiv). The reaction mixture was heated at 80 8C( MW or conventional heating) for the required period of time. The reaction mixture was cooled to RT and diluted with 5mL of as aturated solution of NaHCO 3 .T he aqueous layer was extracted with ethyl acetate (3 10 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 and the solvent removed in vacuo. Purification by column chromatography using silica gel (Hexanes/ethyl acetate 4:1). Characterisation data for isolated indole-Heck products (3, 13-18)a re presented in the Supporting Information.
General protocol for Heck cross-coupling of unprotected halotryptophans with acrylic acid in aqueous conditions:I na0.5-2mLM Wv ial, sodium tetrachloropalladate (1.5 mg, 5 mmol, 10 mol %), with appropriate ligand (TPPTS 6.5 mg or TXPTS: 7.0 mg, 11.5 mmol, 23 mol %) were purged with argon and stirred at RT for 15 min after adding 1mLo fd egassed water/acetonitrile (3:1) mixture, 1mL. Then, the corresponding halo-tryptophan (0.05 mmol) was added together with Na 2 CO 3 (22 mg, 0.2 mmol, 4equiv) followed by addition of the acrylic acid (6 ml, 0.075 mmol, 1.5 equiv). The vial was closed, and the reaction mixture was stirred and heated at 90 8C(MW) for 1hour.A fter completion, the reaction was cooled down to room temperature was diluted with water (2 mL) and acidified (pH = 2-3) using 0.1 m HCl. Solvent was removed under reduced pressure. The desired product was obtained by purification using gradient reversed phase chromatography (C18, 12 g) eluting with MeOH-water (5-95 %gradient).  General protocol for Heck cross-coupling of unprotected halotryptophans with styrene derivatives in aqueous conditions.I na 0.5-2 mL MW vial, sodium tetrachloropalladate (1.5 mg, 10 mmol, 10 mol %), TXPTS (7.0 mg, 23 mmol, 23 mol %) were purged with argon and stirred at RT for 15 min after adding 1mLo fd egassed water/acetonitrile (3:1) mixture. Then, appropriate halo-tryptophan (0.05 mmol) was added together with Na 2 CO 3 (22 mg, 0.2 mmol, 4equiv) followed by addition of the styrene derivative coupling partner (0.075 mmol, 1.5 equiv). The vial was closed and the reaction mixture was stirred and heated at 90 8C( MW) for 2hours. After completion, the reaction cooled down to RT was diluted with water (2 mL) and extracted with diethyl ether (3 2mL) to remove the excess of the alkene coupling partner.T he aqueous layer was acidified (pH = 2-3) using 0.1 m HCl. Solvent was removed under reduced pressure. The desired product was obtained by purification using gradient reversed phase chromatography (C-18, 12 g) eluting with water-MeOH (5-95 %gradient).  Barettin purification from extract [21] Barettin extract was kindly provided by Dr.P aco Cµrdenas, Uppsala University,S weden;2go ff reeze-dried extract was added onto a filter paper in af unnel and rinsed copiously with dichloromethane to remove any lipids from the sample. After rinsing with DCM, the freeze-dried extract was washed thoroughly with 60 %a queous acetonitrile. The washings were checked via LCMS and combined. The sample was concentrated to 1.5 mL.
Barretin extract was purified via RP-HPLC using aP henomenex Luna C18 (5 micron, 250 21.2 mm) with UV detection at 234 nm. The compound was eluted using as hallow linear gradient with starting conditions 95 %s olvent A( 0.1 %f ormic acid in MQ water) to 5% solvent B( ACN) to 40 %s olvent Bo ver 40 min. Over the next 15 min solvent Bw as increased to 95 %, held isocratically for 5min before returning to starting conditions. Barettin (8 mg) eluted with ar etention time of 37 min and was confirmed by LCMS and characterised by NMR. Heck cross-coupling on purified Barettin in aqueous conditions giving 1-(3-((R)-5-(Z)-((6-(4-fluorostyryl)-1H-indol-3-yl)methylene)-3,6-dioxopiperazin-2-yl)propyl)guanidine( 43):As tock solution of catalyst was prepared as follows:s odium tetrachloropalladate (1.5 mg, 5 mmol), TXPTS (7.0 mg, 11.5 mmol, 23 mol %) were purged with argon and stirred at RT for 15 min after adding 1mL of degassed water/acetonitrile (1:1) mixture. Then, in as eparate MW vial barretin (2 mg, 0.005 mmol) was added together with Na 2 CO 3 (2 mg, 0.02 mmol, 4equiv) followed by addition of the 4fluorostyrene (4 mL, 0.15 mmol, 10 equiv) and Pd-catalyst (10 mol % from stock). The vial was closed, and the reaction mixture was stirred and heated at 90 8C( MW) for 2.5 hours. LC-MS analysis showed full conversion. Purification was done via RP-HPLC using a Phenomenex Luna C18 (5 micron, 250 21.20 mm) with UV detection at 330 nm. The compound was eluted using as hallow linear gradient with starting conditions 95 %s olvent A( 0.1 %f ormic acid in MQ water) to 5% solvent B( ACN) to 40 %s olvent Bo ver 40 min. Over the next 15 min solvent Bw as increased to 95 %, held isocratically for 5min before returning to starting conditions. Barettin-Heck derivative eluted with ar etention time of 39 min and was confirmed by LCMS and 1 HNMR. Depicted product was obtained 0.5 mg (21 %isolated yield) as awhite solid. Culture conditions and isolation of pacidamycin Da nd Brpacidamycin D( 44). [8] Starter cultures of engineered strain Streptomyces coelicolor RG1104, with prnA knock-in (performed as previously reported) [3] were obtained by inoculating 150 mL ISP2 medium with 0.3 mL spore suspension (approximately 10 6 -10 7 cfu mL À1 final concentration) and culturing for 24-48 hours at 28 8C, 220 rpm. Starter culture (20 mL) was then added to 0.5 LI SP2 and incubated with shaking at 28 8Cf or 7-8 days. Pacidamycins were extracted from the cell-free broth using 0.05 volumes of XAD-16 resin. The resin was washed with 20 volumes of water and the extract was eluted with 10 volumes of methanol. The solvent was removed in vacuo. The crude extract was then purified by ion-exchange chromatography using a5mL HiTrap SP-FF column (GE Healthcare). After loading, the column was washed with 6volumes 50 mm sodium acetate, pH 3.6. Pacidamycins were eluted with 50 mm sodium acetate in as tepwise gradient from pH 3.6 to pH 5.6. the appropriate pacidamycin-containing fractions were combined and further purified on aL una C18(2) 250 22.10 mm column, initial composition 10 % acetonitrile 90 %R Pb uffer A, held for 2min before reaching 40 % acetonitrile over 40 min compound eluted at around 28 min. Over the next 20 min quick gradient up to 95 %a cetonitrile which was held for 5min before returning to starting conditions. Pacidamycin-D: 1  Heck cross-coupling of Br-pacidamycin Dt ogive 45 To as olution of the purified 7-Br-pacidamycin D( 0.5 mg) in degassed water-acetonitrile (3:1), sodium tetrachloropalladate (10 mol %), TXPTS (23 mol %) (from stock solution in degassed water-acetonitrile (3:1) 10 mm)w ere added followed by Na 2 CO 3 (4 equiv) and 4-fluorostyrene (5 equiv) were added. The vial was closed and the reaction mixture was stirred and heated at 90 8C (MW) for 2hour.A fter completion, the reaction was cooled down to RT was diluted with water (10 mL) and acidified (pH = 2-3) using 0.1 m HCl. The resulting mixture was extracted with ethyl acetate (3 10 mL). Complete conversion was observed by LC-HRMS analysis of the crude reaction mixture. Attempted purification by HPLC was not successful to isolate desired product, which may be due to very low quantities of product 45.