Novel Types of Hypermodified Fluorescent Phyllobilins from Breakdown of Chlorophyll in Senescent Leaves of Grapevine (Vitis vinifera)

Abstract The tetrapyrrolic chlorophyll catabolites (or phyllobilins, PBs) were analyzed in yellow fall leaves of the grape Chardonnay, a common Vitis vinifera white wine cultivar. The major fractions in leaf extracts of V. vinifera, tentatively assigned to PBs, were isolated and their structures elucidated. The dominant fraction is a dioxobilin‐type non‐fluorescent Chl‐catabolite of a previously observed type. Two less polar fluorescent PBs were characterized as a novel dioxobilin‐type fluorescent Chl‐catabolite with a bicyclo‐1′,6′‐glycosyl architecture, and its new fluorescent formyloxobilin‐type analogue. The discovery of persistent hypermodified fluorescent PBs with the architecture of bicyclo‐[17.3.1]‐PBs (bcPBs), suggests the activity of an unknown enzyme that forges the 20‐membered macroring at the tetrapyrrolic core of a fluorescent PB. bcPBs may play specific physiological roles in grapevine plants and represent endogenous anti‐infective agents, as found similarly for other organic bicyclo‐[n.3.1]‐1′,6′‐glycosyl derivatives.

Here, we describe as tudy of the PBs in extracts of naturally senescent leaves of Chardonnay (Vitis vinifera), one of the most important and oldestw hite wine cultivars worldwide. [18] In leaf extracts of Chardonnay plants grown in av ineyard in the province of Bozen (northern Italy), we found both, type Ia nd type II PBs, and discovered two remarkably structured new representatives of "hypermodified" fluorescent PBs, as well. [2d, 12a] The structures of both fluorescent PBs display the exceptional bridging bicyclo [17.3.1]-1',6'-glycopyranosyl architecture, discoveredi nUg-NCC-53. [11f] This finding helps to specify more closely the pathway of the new branch of Chl breakdown to bicyclo [17.3.1]-1',6'-glycopyranosyl PBs (bcPBs) ande ncourages considering ar elevant role of the (fluorescent) bcPBs in the endogenousd efensea gainst fungal and bacterial pathogens in the grapevine leaves.

Results
Leaveso fh ealthy Chardonnay plants, growni na ne xperimental vineyard in Piglon, Laimburg, Italy,d e-greened and developed their characteristic golden yellow in late fall of 2014 (see Figure1). De-greening of healthyl eaves in this region occurs, typically, between the beginning of October and early November and depends on climatic, meteorological and geographical conditions. Yellow,s enescent Chardonnay leaves were collected at the experimental vineyard in November 2014, immediately cooled (external ice packages) for transport to the coldstoragef acility,w here they were stored at À80 8C, until extraction for further analyses (see the Experimental Section). Extracts of the V. vinifera leaves were analyzed by HPLC with UV/ Vis and fluorescenced etection, leadingt ot he provisional identification of eight colorless PBs and of one yellow PB (see Figure 1). The PBs from V. vinifera (Vv-PBs) were tentatively classified as (five) dioxobilin-type non-fluorescent Chl catabolites (DNCCs), as an on-fluorescent( formyloxobilin-type)C hl ca-Scheme1.Structuraloutline of the PaO/phyllobilin pathwayofC hl breakdown, presented with key steps of the C16-stereochemical branchstarting with epi-pFCC.The deformylating enzyme CYP (identified as CYP89A9 in A. thaliana)c onvertsF CCs (fluorescent typeIPBs)i ntoa nalogous DFCCs (fluorescenttype II PBs, the precursors of nonfluorescentDNCCs).
Am ethanolic extract of 600 go fs enescent leaves of V. vinifera,f rom Chardonnay, was separated into fourteen fractions by preparative medium pressure liquid chromatography (MPLC), using the solvent components MeOH and 25 mm aqueous phosphate buffer (pH 7). Twof ractions elutingw ith a nearly 1:1m ixture of the two solvent components contained the major amount of pure Vv-DNCC-51,a ccording to analysis by HPLC. Removal of the solvents of these two combined MPLC-fractions, desalting by the use of SepPak cartridges furnished 60.2 mg (96 mmol) of Vv-DNCC-51.F rom further preparative separation by HPLCo ft hree slightly less polar minor fractions,t wo blue fluorescent phyllobilins and an onfluorescent compound (an NCC) were isolated, furnishing pure samples of 0.  Figure S1). They weret hen further characterized by ESI mass spectra [19] that furnished their molecular formulas (see Figure 3a nd Supporting Information, Figures S2 and S3).   The most polar of the Vv-PBs, named Vv-DNCC-51 (phenomenologically), [13] has previously been detected in extracts of Vvleaves (Pinot noir cultivar), and was suggested to represent a DNCC with the molecular formula C 34 H 40 N 4 O 8 ,b ased on its mass spectrum. [16d] The ESI-MS spectrum of the here isolated Vv-DNCC-51 confirmed the proposed molecular formula (see Figure 3a nd Experimental Section). The structure of Vv-DNCC-51 was fully characterizedh ere, confirming the earlier,t entative, proposal of the chemical constitution of this DNCC.I nterestingly, in the extract of V. vinifera leaves,f our relativelyp olar PB-fractionsw ith UV/Visa bsorption spectra of aD NCC were observed, isomers (epimers) of Vv-DNCC-51,a ccording to their mass spectra, but not characterized further.
In the V. vinifera leaf extract,t he slightly less polar fraction of af luorescent Vv-PB was classified as aD FCC based on its UV/Vis absorption spectrum, andn amed Vv-DFCC-53 (see Figure 4). [17] It showedastrong fluorescence, with an emission maximum at 435 nm, and with an excitation spectrum matching its electronic absorptionp roperties (see Figure 4). Analysis of the mass spectrum of Vv-DFCC-53 revealed ap seudo-molecular ion [M+ +H] + at m/z 777.1 (see Figure 3a nd Experimental Section). This ion is consistent with am olecular formula of C 40 H 48 O 12 N 4 ,s uggestive of an exceptional glycosylated type II PB.
The third fraction was named Vv-FCC-55,a si th ad ar etention time of about 55 min and was an FCC, accordingt oi ts UV/Vis-spectrum (see Figures 1a nd 4). Its strongf luorescence had an emission maximum at 440 nm, with ac haracteristic excitation spectrumo fa nF CC [20] (see Figure4). The ESI mass spectrumo fVv-FCC-55 revealed ap seudo-moleculari on [M+ +H] + at m/z 789.1, consistent with am olecular formula of C 41 H 48 O 12 N 4 (see Figure 3a nd Experimental Section). Hence, Vv-FCC-55 was indicated to contain one carbon atom more per molecule than Vv-DFCC-53,suggesting their close structural relationship.
The ESI mass spectrum of the less polar fraction of Vv-NCC-57,classifieda sanN CC by its UV/Vis-spectrum (see Experimental Section and Supporting Information, Figure S1) showed a pseudo-molecular ion [M+ +H] + at m/z 645.3 (see Experimental Sectiona nd Supporting Information, Figure S2), consistent with am olecular formula of C 34 H 40 N 4 O 8. This indicated Vv-NCC-57 to contain one carbon atom more per molecule than Vv-DNCC-51.H PLC-analysis,i ncluding the co-injection of Vv-NCC-57 and of Cj-NCC-1( see Experimental Section and Supporting Information, FigureS4), confirmed the identityo fVv-NCC-57 with an abundant NCC of the "epi"-series, first obtained from senescent leaves of Cercidiphyllum japonicum, named Cj-NCC-1. [4b, 6a] This established the C16 "epi"-configuration of Vv-NCC-57 and also indicated the commonC 16-configuration as "epi" for the other colorless Vv-PBs.
As econd set of 500 MHz NMR spectra from as olution of Vv-DFCC-53 in CD 3 CN exhibited the full signal of the exchange labile H-atom at C8 2 of ring E( see Supporting Information, Ta ble S4 and Figure S8). The correlation of HC8 2 to HC5' of the glucopyranose moiety in the NOE-spectrum provided evidence for the closem utual positioning of these two units in space. 1 H, 13 Ch eteronuclear spectra (in both solvents) provided as et of single bond correlations (HSQC) and multi-bond correlations (HMBC)t hat established the two sites of covalenta ttachment of ap yranose-unit to O3 3 of ring Aa nd O12 4 of the propionate substituent of the PB core of Vv-DFCC-53.T he 1 Ha nd 13 C chemicals hifts at the methylene group H 2 C3 2 were also consistent with an attached peripheralsugar substituent, as were the 13 Cs hifts of C5'and C6' of the sugar moiety with an ester linkage at C6' (see Figure 6). The bridging sugar-moiety of Vv-DFCC-53 was( further) identified as a1 'b-glycopyranosylg roup by comparison of the chemical shifts of its 1 Ha nd 13 Ca toms with those of the sugar moietyo fUg-NCC-53. [11f] Based on the furthers tereochemical characterization (see below), Vv-DFCC-53 is deduced to be a4 R, Likewise, a7 00 MHz 1 HNMR of Vv-FCC-55 (in CD 3 OD, at 25 8C) showed resonances of af ormylg roup and of av inyl group, the singlet of am ethyl ester function, and three singlets and ad oublet, characteristic of the four other methyl groups of an FCC (see Supporting Information, Figure S7). The signals of all 41 exchange stable H-atoms of Vv-FCC-55 were found and assigned,a sw ere 37 of the 41 C-atoms of this PB (see Experimental Section and Supporting Information, Ta ble S3). As econd set of 600 MHz NMR spectraf rom as olution of Vv-FCC-55 in CD 3 CN provided complementary data including those of the exchange labile H-atom at C8 2 of ring E (see Supporting Information, Table S5 and Figure S9). The NOEcorrelation of HC8 2 to HC5' of the glucopyranose moiety in the spectrum of Vv-FCC-55 provided evidencef or the close mutual positioning of these two units and for the indicated macrocyclic structure. 1 H, 1 Hh omonuclear correlations( ROESY-spectra) and 1 H, 13 C heteronuclear correlations (HSQC and HMBC spectra) in CD 3 OD solution (see Figure 7) and in CD 3 CN (see Supporting Information, Ta ble S5 and Figure S9) indicated an attachment of a sugar moiety at O3 3 .T he shifts of the 1 Ha nd 13 Cs ignals for the C3 2 methylene group were also consistent with the presence of ag lycosidic substituent at O3 3 .T he chemical shifts of C12 3 at ring Cw ere consistentw ith the presence of ap ropionyl ester functionality and indicated al ink to the primary oxygen    stereo-projection of ac alculated conformer). The mutuala rrangemento ft he glucosea nd tetrapyrrole moieties are in line, qualitatively,w ith NOE-datad erived from homonuclear ROESY spectra (see Supporting Information, Figure S9 and S13) with a calculated distance of 2.7 between HC8 2 (of the FCC moiety) and the glucopyranose HC5'.F or aq ualitative comparison, the structureo fUg-NCC-53, derived from the molecular dynamics study, [11f] was also optimized computationally (see Supporting Information, Figure S19), indicating ah igher stability,b y around7 5kJmol À1 ,o fUg-NCC-53, compared to its fluorescent isomer, Vv-FCC-55,i nt he respective calculated conformations.
The (gas phase) structures of both of the C4-stereoisomers of Vv-DFCC-53 were also modelled, which differed in the configuration at C4. The sugar moiety was again calculated as sitting "atop" of the B/E-ring section, positioning the glucose HC5' at ad istance of 2.6 from HC8 2 of the bcPB, orienting ring An early orthogonal to the B/E-ringp lane,a nd presenting the C2ÀC3-periphery to the top side of both isomeric molecules. Hence, HC4 is pointing towards the glucopyranosyl group (i.e. "endo") in the R-epimer,b ut in the opposite direction (i.e. "exo") in the S-epimer (see Figure 9a nd Supporting Information, Figures S15-S18).N OE correlations between HC4 and H 2 C3 2 are observed in ROESY spectra of Vv-DFCC-53,c ompatible with an "endo"p osition of HC4, as seen in the model of the R-epimer.I nterestingly,t he quantum chemical studies also revealed R-Vv-DFCC-53 to be slightly more stable in the gas phase than its S-epimer (1.7 kJ mol À1 or 10.4 kJ mol À1 ,w ithout or with incorporation of dispersion interactions, respectively).

Discussion and Outlook
Grapevine (Vitis vinifera)i sawidespread and prehistoric domestic agricultural plant. It is an exceedingly valuable crop worldwide, [22] with Chardonnayb eing one of the most important white wine cultivars. Beside their use forw ine production, grapes can be sold fresh on markets and in stores, are the basis for juice production,o rc an be dried as raisins. [18,23] Additionally,t he use of grapevine leaves is populari nd ifferent cuisines, especially in Greek, oriental and Asian cooking( see, e.g. [24]). In fall, grapevine leaves of white wine cultivars undergoa color change to bright orange, as ign of the seasonal Chl breakdown and leaf senescence. [2c] As shownh ere with the example of leaves of aC hardonnay cultivar,i nn aturally senescent leaves of grapevine (V. vinifera) type Ia nd type II phyllobilins (PBs) accumulate, [2d, 3] as was also found in other higher plants, recently. [16a, 25] However, the structures of some Vv-PBs reveal au nique pattern of PB-modifications. Twon ovel types of fluorescent PBs, Vv-DFCC-53 and Vv-FCC-55,i np articular,w ered iscovered here and found to belong to the exceptionalc lass of the bicyclo[17.3.1]-phyllobilins (bcPBs) with a1 'b,6'-d-glycopyranosyl bridgel inking O12 4 and O3 3 .F luorescent bcPBs are as pecial variant of the "hypermodified" FCCs (hmFCCs) that are made persistentb ya ne ster modification of their propionate function. [6b] The two fluorescent bcPBs show the amazing structuralf eaturesa ctually discoveredw itht he non-fluorescent analogue Ug-NCC-53, isolated from senescent leaves of the wych elm. [11f] The sugarbridged bicyclo [17.3.1]-architecture of Ug-NCC-53 imposed a rather rigid framework onto the flexible core structure of this NCC, giving it extraordinary 3D-structural features. The structure of Ug-NCC-53 encouraged to consider relevant physiological roles for this bcNCC and to look out for convincing insights into its biosynthetic formation during Chl breakdown. [11f] Indeed, the presence of two NCCs glycosylated at their O3 3 in leaf extracts of the elm tree suggested the occurrence of the corresponding FCCs as catabolic precursors. In spite of this, the specific pathway to the bcNCC Ug-NCC-53 has remained obscure. [11f] The existence of the two fluorescent bcPBs, Vv-DFCC-53 and Vv-FCC-55,d isplaying the amazing bicyclo[17.3.1]-architecture with a1 '6'-glycopyranosyl-moiety bridging O12 4 and O3 3 in the two novel Vv-PBs, contrasts the presumption that the existence of such bridgesw ould be restricted to the bcNCCs, such as Ug-NCC-53. [11f] Based on computational modelling, this NCC appeared to be ar ather unstrained and stable bicyclo[17.3.1]glycosidicm olecule, by virtue of the adaptive structure of its flexible phyllobilaneb ackbone. [11f] Strikingly,a ss hown here, fluorescent bcPBs, with an unsaturated linkage between rings B/E and C, also exist and are generated in the course of the PaO/phyllobilinp athway of Chl breakdown in grapevine leaves.
Indeed,a ne ntirelya lternative biosynthetic formation of Vv-DFCC-53 may proceed directly by oxidative deformylationo f its FCC-precursor,t he bicyclo-glycosylated Vv-FCC-55.T his scenario would require the corresponding structural tolerance of the deformylating cytochrome P450 enzyme (CYP89A9 in A. thaliana) [16a] for the FCC-substrates, which may not be likely, but hasn ot been tested yet. Vv-DFCC-53 represents as ingle stereoisomer,t entatively assigned here as the 4R-epimer,f rom comparison of the calculateds tructures of 4S-a nd 4R-epimers of the DFCC with experimental NOE-correlations.
Glucosylations, as observed in various NCCs, [2d, 5] were first interpreted as at ypicalr esult of "secondary" metabolism in the context of the "Chl-detoxification"h ypothesis of the Chl breakdown path. [29] However, the formation of hmFCCs in ripening fruit ands enescentl eaves appeared to be ar ational consequenceo fadeliberate "biosynthetic" effort of some plants, with the purpose of generating luminescent pigments. [12,14] The discovery of the fluorescent bcPBs Vv-FCC-55 and Vv-DFCC-53 in grapevine leaves, likewise, suggestsaspecial "biosynthetic" input in generating such "persistent" bcPBs,r ationalized, again,b yaphysiological benefit in the leaves from such bcPBs.
So far,t he 1'-b-d-glycosyl-transferases involved in the formation of the FCC-glucosides, such as O3 3 -b-d-Glc-pFCC, [26] are unknown, [2a] as are plant acyltransferases to sugars [30] of at ype required for the assembly of hmFCCs [2d] from an activated FCC. Hence, both types of enzymes remaint ob ei dentified in higher plants. Likewise, unknown are the plant enzymes capable of forging the 20-memberedm acro-ring in the bicyclo-1',6'-glycosyl-architecture of bcPBs by setting up the second one of the two conjugations of the glycopyranosyll inker with the tetrapyrrolic core of an FCC. Along with the current evidence about the location of typical Chl-catabolic enzymes, [2c] not only the still elusivee nzymes that introduce sugar units in fluorescent PBs, but also those closing the bicyclo-1',6'-glycosyl-macroring, would be proposed to be active cytosolic proteins. [2a] This hypothesis would exclude ap ath to the bicyclo-1',6'-glycosyl-structure at the stage of an NCC, i.e.,a fter import into the vacuole. [11f] Clearly, the here reported discovery of the amazing fluorescent bcPBs poses intriguing new questions with respect to the biosynthetic paths to these uniqueC hl-catabolites with ab icyclo-1',6'-glycosyl-macroring.
Natural heterocyclic products displaying as ugar bridgedb icyclo-[n.3.1]-structurew ereu nknown before the discovery of Ug-NCC-53. [11f] Typical sugar appendages in natural products are bound as terminal1 '-glycosides or in al inear oligosaccharide topology. [30b, 31] However,t he biological toolbox with sugar appendages is far from being explored, and Nature's capacity for "natural-product glycoengineering" is enormous. [30b] A range of natural, and semisynthetic non-pyrrolic organic compounds exhibit the exceptional1 ,6-glycopyranosyl-bridged macrocyclic bicyclo-[n.3.1]-structure and are the target of considerable interestf rom biological and pharmaceutical points of view. [11f,32] Indeed,a mong such 1,6-glycopyranose-bridged organic compounds, also classified as ansaglycosides, [32d] figure inhibitors of cell growth, [33] as wella sc ompounds with antifungal, [34] antibacterial [32a, 34b, 35] and antiviral effects. [32b, 36] The exceptional bcPBs in grapevine leaves may be surmised to play crucial (however,s till elusive) physiological roles, both in plants and in humans. Important experimentale videncea long these lines comesf rom the recently identified PBs in pathogenically de-greened apple and apricot leaves, [37] suggesting ar ole for PBs in the interaction of the plant host with bacterial or fungal pathogens, either as part of the plant's immune response [38] or the pathogen's virulence strategy.L ikewise, the possible health effects of such intriguing naturalp roducts as componentso f our daily nutrition are also an attractive, but stillu nexplored area of research. [2d] Complexm acrocyclic skeletons, like that of bcPBs, are af eature of physiologicallya ctive natural products, and are recognizeda se volutionary privileged structures in modern drug design approaches. [32c, 39] Twoo ft he colorless phyllobilins in naturallys enescent, golden-yellow leaves of grapevine (V. vinifera)w ere characterized as an FCC and aD FCC that belongt ot he wider class of the hypermodified fluorescent PBs and represent the specific subtype of the bcPBs with ab icyclo [17.3.1]-glycosyl structure (see Scheme 3). The biosynthetic generation of this structural feature of bcPBs is puzzling, and is ac hallenge to be pursued further. Comparison of the NOE correlationso bserved in ROESY spectra of Vv-DFCC-53 provides evidencei ns upport of the R-configurationa tC 4, leadingt oaf irst tentative stereochemicala ssignment of at ype II PB at the new asymmetric C4. It will be of interestt of ind furthers upport for the (general) validity of this stereochemical assignment in type II PBs.
The discovery of bcPBs,f urthermore, not only enlarges the portfolioo ft he knownP Bs, and their structural diversity,b ut may also open an ew chapter in the search for the still elusive roles of Chl catabolites in senescent leaves and other plant organs. [2a,d, 4d, 12b] Persistent blue fluorescent PBs, such as those now found in grapevinel eaves, are (potential) endogenous sensitizers for the formation of singlet oxygen. [20] They also contribute as natural optical brighteners to theo ptical appearance of the leaves, [14] af actor considered relevant in bio-communication. [40] The exceptionals tructures of bcPBs may also provide an ew drive to the quest foru ncovering relevant pharmacological effects of the abundant, and often, uniquelys tructured PBs.

Experimental Section
Plant material Senescent, yellow colored leaves were collected on November 14th, 2014 from healthy Chardonnay grapevine plants in an experimental vineyard ("Piglon"), at Laimburg Research Centre (Pfatten/ Vadena, South Tyrol, Italy). The grapevines, planted in 2006, were grown on aG uyot training system and managed according to the integrated production guidelines. The leaves were transported on ice to the laboratory,i mmediately frozen to À80 8C, and transported frozen to Innsbruck, where they were stored at À80 8Cu ntil analyses.

Leaf extraction and isolation of Vv-PBs
As ample of 600 g( wet weight) of yellow Chardonnay leaves (collected November 14th, 2014), frozen at À80 8C, was crushed cold to ap owder with am echanical mixer,suspended in 500 mL of cold MeOH and again mixed for two more min. The suspension was filtered through ac oarse glass filter and the filter cake was washed with 100 mL MeOH. The combined filtrates were stored at 4 8C. The remaining filter cake was re-suspended in 500 mL of MeOH by mixing mechanically for 2min and filtered again. This operation was repeated once more. The three filtrates were combined (about 1500 mL) and solvents were removed under vacuum and at < 30 8Ct oaresidual volume of roughly 100 mL by the use of ar otatory evaporator.T he raw isolate was frozen at À20 8Cf or overnight storage. Subsequently it was mixed with 1Lof 25 mm aqueous potassium phosphate buffer (pH 5.2) and transferred into a separation funnel, to be extracted by four sequential batches of MeCl 2 (1 L, 750 mL, 500 mL and 500 mL). The combined organic phases were dried by passage through alarge plug of dried cotton wool and the solvents were removed completely under vacuum by the use of ar otatory evaporator.T he residue was dissolved in 20 mL of MeOH and 80 mL of 25 mm aqueous potassium phosphate buffer (pH 7) were added. Ay ellow powder formed, which was removed by centrifugation. The clear supernatant was stored overnight at À80 8C. The sample was applied to the column of the MPLC-system and was separated into 20 fractions, which were analyzed by HPLC. Fractions 6a nd 7c ontained pure Vv-DNCC-51 and were combined (90 mL, in total) and concentrated to about 50 mL containing "pre-purified" Vv-DNCC-51.F ractions 10 and 11,w hich contained impure Vv-DFCC-53 (in about 40 mL solvent, each), were also concentrated under vacuum and at < 30 8Ct oaresidual volume of roughly 20 mL by the use of ar otatory evaporator,d esalted (1 gS epPak cartridge) and stored frozen at À20 8C( as "raw" Vv-DFCC-53)f or further purification by preparative HPLC (see below). Fractions 12 and 13, which contained impure Vv-FCC-55 in about 40 mL solvent (each), were also concentrated under vacuum and at < 30 8Ct oar esidual volume of roughly 20 mL using ar otatory evaporator and desalted on a1gS epPak cartridge. Solvents were removed to furnish two samples of "raw" Vv-FCC-55 for further purification by preparative HPLC (see below). Isolation of Vv-DNCC-51:t he sample of "pre-purified" Vv-DNCC-51 was used in two similarly sized batches, which were each desalted using a5gS epPak C18-cartrige. Solvents were removed under vacuum and at < 30 8Ct oar esidual volume of roughly 2mLb y the use of ar otatory evaporator.T he two residual samples were combined and frozen with liq. N 2 and lyophilized overnight, furnishing 60.2 mg of Vv-DNCC-51 as an off-white powder.
Isolation of Vv-DFCC-53:t he two desalted MPLC-fractions were combined and dissolved in roughly 0.5 mL of a4:1 (v/v) mixture of 25 mm aqueous phosphate buffer (pH 7) and MeOH for separation by semi-preparative HPLC in two similarly sized batches. From each run the main fraction w as collected and analyzed by HPLC for purity.T he combined purified samples were desalted by the use of a1gSepPak cartridge. Solvents were removed using ar otatory evaporator under vacuum and at < 30 8C. The residual sample of Vv-DFCC-53 was dissolved in 20 mL of MeOH and analyzed quantitatively as 0.38 mg by recording its UV/Vis spectrum. Solvents were removed and the residue of Vv-DFCC-53 was dried using high vacuum, for storage at À80 8Cf or further analysis. Isolation of Vv-FCC-55:T he two samples of "raw" Vv-FCC-55 were dissolved in roughly 0.5 mL each of a4 :1 (v/v) mixture of 25 mm aqueous phosphate buffer (pH 7) and MeOH and separated by semi-preparative HPLC. The fractions collected were analysed by HPLC for content. From each run the main fraction with pure Vv-FCC-55 was desalted by the use of a1gSepPak cartridge. Solvents were removed using ar otatory evaporator under vacuum and at < 30 8C. The dried samples of Vv-FCC-55 were dissolved in 20 mL of MeOH each and analysed quantitatively as 0.26 mg (from MPLCfraction 11)a nd 0.39 mg (from MPLC-fraction 12) by recording UV/ Viss pectra. The samples of Vv-FCC-55 were combined, solvent was removed and the residual samples of Vv-FCC-55 were dried and stored frozen at À80 8Cf or further analysis.
Isolation of Vv-NCC-57 and of Vv-DYCC-63:f rom the semi-preparative HPLC experiments fractions were collected, combined and desalted that had HPLC-retention times and UV/Vis-spectral properties of Vv-NCC-57 or of Vv-DYCC-63.T he resulting samples of Vv-NCC-57 and Vv-DYCC-63 were each dissolved in 5mLo fM eOH and analysed quantitatively by recording their UV/Vis-spectra, indicating 1.25 mmol (0.78 mg) of Vv-NCC-57 and 0.95 mmol (0.59 mg) of Vv-DYCC-63.S olvents were removed and the residual samples of Vv-NCC-57 and Vv-DYCC-63 were dried and stored frozen at À80 8Cf or mass spectrometric analysis.

Computational methodology
The initial structure of Vv-FCC-55 was developed from the previously published NCC [11f] gas phase structure and modified to be consistent with the stereochemistry derived from NMR (NOE) data using GaussView 6.0 [42] and Schrçdinger's Maestro [43] tool. Subsequently,t he initial Vv-FCC-55 conformer was structurally optimized in the gas phase using Density Functional Theory.T he BP86 [44] density functional was employed in combination with the resolutionof-identity technique [45] and the def2-TZVP basis set. [46] Empirical dispersion corrections of the Grimme type were also tested [47] but had little effect on the resulting structures (see overlay in Figure S14) The gas phase structures of the two Vv-DFCC-53 C4-stereoisomers were generated from the optimized Vv-FCC-55 structure. All calculations were performed with Turbomole [48] and structures were visualized with PyMol. [49] Spectroanalytical data