Andrzej Wolan

Justyna A Kowalska-Six

Holisoa Rajerison

Michèle Césario

Marie Cordier

Yvan Six
email: yvan.six@polytechnique.edu

,3-Dipolar cycloadditions with azomethine ylide species generated from aminocyclopropanes

Keywords: Cyclopropane, 1,3-Dipolar Cycloaddition, Azomethine Ylide, Microwave-assisted Synthesis, Pyrrolizidine, Titanium

Two types of bicyclic N-cyclopropyl glycine ester derivatives have been prepared and put under scrutiny as possible precursors of azomethine ylides. The results demonstrate that they can indeed participate in 1,3-dipolar cycloaddition reactions with dipolarophiles, as illustrated in the cases of phenyl vinyl sulfone, N-phenylmaleimide, diethyl fumarate and diethyl maleate. The relative configurations of the major diastereoisomers produced are consistent with the predicted generation of azomethine ylide species, reacting in concerted cycloaddition processes. This unprecedented way of generating such 1,3-dipoles provides access to functionalised pyrrolizidines and pyrrolidines, that would be difficult to make directly by more classic methods. It was also found that using phenyl vinyl sulfone or N-phenylmaleimide as the dipolarophile reactant, a domino nucleophilic conjugate addition / 1,3-dipolar cycloaddition process may operate competitively.

M A N U S C R I P T A C C E P T E D

1. Introduction 1,3-Dipolar cycloaddition reactions constitute a general and versatile tool for the synthesis of fivemembered ring heterocycles. 1 Among the various 1,3-dipoles that can be used, azomethine ylides 1 have led to a number of interesting applications, as they can provide access to diversely substituted pyrrolidines, as well as to oxazolidines and imidazolidines. 2 A number of methods have been developed for the generation of these dipoles, among which one of the most popular relies on the deprotonation of iminium species 2 featuring acidic hydrogens at the α position relative to the nitrogen atom (Scheme 1, equation 1). 3 Since the protonation of bicyclic aminocyclopropanes 3 is an efficient and original route for the generation of iminium cations 4 (equation 2), 4 we envisioned that the derivatives 5 could be suitable precursors to cyclic azomethine ylides 6 (equation 3). Scheme 1. Strategy for the generation of azomethine ylides from aminocyclopropanes. EWG = electron-withdrawing group. (eq. 2)

(eq. 3)

H + H + + + azomethine ylide H EWG EWG H R 1 + N R 2 R 1 3 4 5 6 N H R 1 N R 2 H R 1 + 2 1 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT -3 -

Results and discussion

Synthesis of the substrates

For the purpose of this study, aminocyclopropanes 5a and 5b were readily prepared using an intramolecular Kulinkovich-de Meijere reaction as the key cyclopropanation step (Scheme 2). 5,6,7 Although carboxylic esters are known to be more reactive than amides towards diisopropyloxytitanacyclopropane complexes, 8 this reaction proceeded with remarkably good chemoselectivity from 11 and 12 and no cyclopropanol resulting from an intramolecular Kulinkovich reaction was formed in significant amount. The steric effect exercised by the tert-butyl group, as well as a kinetic effect favouring the formation of a five-membered rather than a six-membered ring may reasonably account for this result.

Scheme 2. Preparation of aminocyclopropanes substrates 5a and 5b.

Cycloaddition reactions with phenyl vinyl sulfone and N-phenylmaleimide

Initial cycloaddition experiments were conducted with aminocyclopropane 5a under standard thermal conditions, with two equivalents of phenyl vinyl sulfone or N-phenylmaleimide as the dipolarophile reactant. In both cases, rather complex mixtures of products were generated: the predicted cycloadducts 13 and 15 were formed with low to moderate diastereoselectivity, together with the unexpected by-products 14 and 16 having incorporated two molecules of the dipolarophile reactant (Scheme 3). The formation of these compounds, also formed as mixtures of diastereoisomers, can be rationalised by invoking an equilibrium between the azomethine ylide intermediate 6a and an endocyclic enamine 17a. 9 The latter would undergo a conjugate addition -4 -reaction / 1,3-dipolar cycloaddition sequence, 10 as illustrated in Scheme 4 in the particular case of phenyl vinyl sulfone, with the generation of a highly substituted azomethine ylide 18a.

Scheme 3. Thermal reactions of 5a with two equivalents of phenyl vinyl sulfone or Nphenylmaleimide (isolated yields).

Scheme 4. Proposed mechanism for the formation of compounds 13 and 14.

For these experiments and the subsequent studies, analysis of the crude reaction products by HPLC/MS provided valuable information with respect to chemoselectivity: direct 1,3-dipolar cycloaddition vs conjugate addition-1,3-dipolar cycloaddition, as well as an assessment of the distribution of the diastereoisomers for both types of products. 11 A typical TIC chromatogram, in the case of the reactions of 5a with N-phenylmaleimide, is presented in Figure 1. The major product 15a is visible, at t = 10.767 minutes, along with 3 other diastereoisomers. Nine diastereoisomers of compound 16 also appear clearly on this chromatogram. The chemo-and diastereo-selectivity of the reaction is thus less than satisfactory. 12 Nonetheless, given the fact that 8 diastereoisomers of the adduct 15 and 16 diastereoisomers of the by-product 16 can in principle be formed, it is worth noting that 15a was comparatively easy to purify and useful amounts of this compound could be isolated after a single flash chromatography. 13 An X-ray structure of this cycloadduct is presented in Figure 2. 14 The relative configuration of its chiral centres is consistent with a W-shaped conformation of the azomethine ylide 6a and with an endo approach of the dipolarophile, if one assumes the cycloaddition to be a concerted process (vide infra). 2d The relative configurations of other purified individual diastereoisomers of compounds 13-16 were tentatively assigned on the basis of NOESY 2D NMR experiments. 13 The influence of several reaction parameters on the product distribution was briefly examined (Table 1). The results of this short study show that the use of only one equivalent of dipolarophile, rather than two, favour the formation of the simple 1,3-dipolar cycloaddition adducts (entry 3 vs entry 1 and entry 4 vs entry 2). However, introduction of phenyl vinyl sulfone in small portions of 0.1 equivalent did not result in the more selective production of the monosulfone adducts 13 (entries 5 and 6 vs entries 3 and 4). It is also interesting to note that the choice of N-phenylmaleimide as the dipolarophile led to the production of comparatively less by-product resulting from conjugate

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addition-1,3-dipolar cycloaddition (entry 8 vs entry 4 and entry 9 vs entry 7). The use of paratoluenesulfonic acid (pTSA) reduced the reaction times by increasing the rate of the protonation of the substrate 5a 15 but also appeared to slightly favour the production of the by-products 14 and 16 (entry 2 vs entry 1, entry 4 vs entry 3, entry 6 vs entry 5 and entry 8 vs entry 9). With this respect, heating the reaction mixtures in a microwave reactor proved very interesting: 15 the reactions were complete after 5 to 10 minutes only, with a better selectivity in favour of the simple cycloaddition adducts (entry 7 vs entries 3 and 4; entry 9 vs entry 8).

Cycloaddition reactions with diethyl fumarate and diethyl maleate, under microwave irradiation

We next turned our attention to the use of diethyl fumarate and diethyl maleate as the dipolarophiles, with microwave heating. The corresponding reactions proved to be much more selective. Indeed, no

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by-products arising from competitive Michael addition were observed and we thus elected reaction conditions involving the presence of 2 mol% of pTSA, ensuring full conversion of 5a (Scheme 5). While all the diastereoisomers 19a-d isolated after the reaction with diethyl fumarate had retained the trans relative configuration of the ethyl ester groups, the reaction with diethyl maleate led to the formation of the new diastereoisomers 20a and 20b, with a cis relative configuration of the same groups. This provides strong support for a concerted cycloaddition process. However, the fact that diastereoisomers 19a and 19d, which had been produced in the reaction with diethyl fumarate, were isolated in significant amounts after the reaction with diethyl maleate as well, did not rule out the possibility of a mechanism operating partly in a stepwise fashion. To get further insight into this problem, a reaction was run in the presence of excess amounts of geometrically pure diethyl maleate (two equivalents). Analysis of the crude product revealed the presence of a mixture of unreacted diethyl maleate and diethyl fumarate, thereby demonstrating partial isomerisation of diethyl maleate into diethyl fumarate under the reaction conditions, which is sufficient to explain the result observed. 13 Accordingly, one can notice that once again, the relative configurations of all the products are consistent with a W-shaped conformation of the azomethine ylide 6a, as drawn in Scheme 4.

Scheme 5. Cycloadditions of 5a with diethyl fumarate and diethyl maleate.

Further experiments were performed with the n-propyl aminocyclopropane derivative 5b, leading to the production of compounds 21-22 with improved selectivities, as compared with the reactions of 5a (Scheme 6).

CO 2 Et EtO 2 C N tBuO 2 C 5a + pTSA (2 mol%) PhCl, 250 W W 140 °C, 10 min tBuO 2 C N EtO 2 C EtO 2 C + tBuO 2 C N EtO 2 C EtO 2 C + tBuO 2 C N EtO 2 C EtO 2 C + tBuO 2 C N EtO 2 C EtO 2 C 50% 19a 12% 19b 13% 19c 11% 19d N tBuO 2 C 5a + pTSA (2 mol%) PhCl, 250 W W 140 °C, 10 min tBuO 2 C N EtO 2 C EtO 2 C + tBuO 2 C N EtO 2 C EtO 2 C + + tBuO 2 C N EtO 2 C EtO 2 C 33% 20a 14% 20b 17% 19a 3% 19d EtO 2 C EtO 2 C tBuO 2 C N EtO 2 C EtO 2 C M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT -8 - Scheme 6.
Cycloadditions of 5b with diethyl fumarate and diethyl maleate.

Possibilities of extension

Finally, preliminary studies have been undertaken with a different kind of bicyclic aminocyclopropane substrate: compound 23, synthesised by the method reported by P. Bertus and J. Szymoniak. 16 This substrate reacted with diethyl fumarate to produce the expected substituted pyrrolidine 24 (Scheme 7), which is promising with respect to the generality of this new mode of generation of azomethine ylide species. Significant differences were observed, as compared with the results obtained with 5a and 5b: firstly, higher temperature (180 °C) had to be applied. Indeed, incomplete conversion of 23 was observed when microwave irradiation was carried out at 140 °C for 10 minutes. Secondly and more interestingly, the triester cycloadduct 24 was obtained as a single diastereoisomer. The relative configuration of its chiral centres, established by X-ray crystallography of the corresponding hydrochloride salt 24.HCl (Figure 3), 14 is again in agreement with a concerted 1,3-dipolar cycloaddition. Indeed, the two diethyl ester groups are in trans relative configuration and the relative configuration of the other chiral centres is consistent with the azomethine ylide intermediate 25 in the conformation displayed in Scheme 7. Moreover, it is interesting to note that the relative configuration of the three ester substituents is identical to the one observed with 19a and 21a, the major cycloadducts of 5a and 5b with diethyl fumarate (Schemes 5 and6).

Conclusions

In summary, our results demonstrate that bicyclic aminocyclopropane derivatives that are substituted with a 2-alkoxy-2-oxoethyl group can undergo rearrangement into azomethine ylide species under thermal conditions. This represents an unprecedented route to these reactive 1,3-dipoles, complementary to classic methods, with an access to differently functionalised molecules. In particular, the substrates 5a, 5b and 23 can be used as precursors of the corresponding ylide species 6a, 6b and 25 and participate in 1,3-dipolar cycloaddition reactions with various dipolarophiles to produce highly substituted pyrrolizidine skeletons. Application of microwave heating conditions has allowed us to reduce the reaction times to a few minutes and to minimise a competitive process that is sometimes observed and that leads to more complex molecules having incorporated two units of the dipolarophile.

The potential of these cycloaddition reactions in synthetic applications is particularly relevant in the field of pyrrolizidine natural product synthesis. 17 It is worthy of note that our method provides direct access to pyrrolizidine skeletons having an alkyl substituent at the ring junction carbon atom. In contrast, to the best of our knowledge, in all the other literature examples of pyrrolizidine cycloadducts, this substituent, if present, is an electron withdrawing group (typically an ester group). 18,19 It is worth pointing out that several pyrrolizidine alkaloids of interest have an alkyl substituent at this position, for instance (+)-NP25302, 20 caldaphnidine M 21 and (-)-penibruguieramine A 22 (Figure 4). In spite of the rather low diastereoselectivities that are often observed in the presently reported cycloaddition reactions, there are several instances where the major diastereoisomers of the products can be isolated in useful yields (up to 65%). It is anticipated that much more selective reactions can be designed on the basis of these first results, for instance by making the cycloaddition process intramolecular. Results of new studies carried out along this idea will be reported in due course.

Experimental

General information

THF was purified using a PureSolv solvent purification system (Innovative Technology Inc.). Analytical grade chlorobenzene was purchased from SDS and used as such. Cyclopentylmagnesium chloride solution in diethyl ether was purchased from Sigma-Aldrich or Fluka and titrated monthly according to a previously reported method. 23 Titanium (IV) iso-propoxide (VERTEC ® TIPT) was purchased from Alfa Aesar, distilled under reduced pressure and stored under argon for several months. The microwave-promoted experiments were run using a CEM Discover Microwave Synthesis System with the temperature and time parameters indicated; the reaction vessels were not flushed with an inert gas. All other reactions were carried out under nitrogen or argon. The temperatures mentioned are the temperatures of the cold baths or the oil baths used. Concentration under reduced pressure was carried out using rotary evaporators at 40 °C. Flash column chromatography was performed on SDS Chromagel silica gel 60 (35-70 µm) or on VWR Chemicals or Merck silica gel 60 (40-63 µm). Petroleum ether refers to the 40-60 °C fraction. NMR spectra were recorded with AVANCE 300 ( 1 H at 300 MHz, 13 C at 75.5 MHz) or AVANCE 400 ( 1 H at 400.2 MHz, 13 C at 100.6 MHz) Bruker spectrometers. Chemical shifts δ are given in ppm, referenced to the peak of tetramethylsilane defined at δ = 0.00 ( 1 H NMR), or the solvent peak of CDCl 3 , defined at δ = 77.0 ( 13 C NMR). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quadruplet, quint = quintuplet, sext = sextuplet, sept = septuplet, m = multiplet, br = broad. Coupling constants J are given in Hz and are rounded to the closest multiple of 0.5. Infrared spectra were recorded with a Perkin-Elmer BX or a Perkin-Elmer 2000 FT-IR spectrometer. Melting points were determined using a Büchi 535 apparatus and were not corrected. Mass spectra were obtained using

Synthesis of 5a

A solution of but-3-en-1-yl methanesulfonate 8 24 (1.00 equiv, 95.2 mmol, 14.3 g) in a mixture of MeOH (95 mL) and 32% NH 3 aqueous solution (95 mL) was vigorously stirred at 20 °C for 3.5 days. The solvents were then removed under reduced pressure to afford a white solid (14. 7 g). Analysis of the crude product by 1 H NMR spectroscopy showed that it contained but-3-en-1ammonium methanesulfonate 9 as the major component, in an estimated amount of 10.4 g (61.9 mmol, 65%). 9: 1 H NMR (CD 3 OD, 300 MHz): δ 2.42 (2 H, tdt, J 7.5, 7.0, 1.5), 2.70 (3 H, s), 3.01 (2 H, t, J 7.5), 4.85 (3 H, br s), 5.14-5.29 (2 H, m), 5.82 (1 H, ddt, J 17.5, 10.0, 7.0).

Crude product of the previous step (1.14 equiv, assumed 47.4 mmol of mixture of ammonium salts, 7.32 g), tert-butyl 2-chloroacetate (1.00 equiv, 41.6 mmol, 5.95 mL) and potassium carbonate (2.00 equiv, 83.2 mmol, 11.5 g) were stirred in MeCN (0.20 L) at reflux for 1 h 30 min. After cooling, the inorganic salts were filtered off and rinsed with MeCN (50 mL) and Et 2 O (50 mL). The combined organic solutions were concentrated under reduced pressure to give crude tert-butyl 2-(but-3-en-1ylamino)acetate 10 as an oil (4.43 g) that was engaged in the next step without purification. 10: Pale yellow oil. IR (neat): ν 2977 (w), 2931 (w), 1731 (s, C=O), 1367 (m), 1223 (m), 1146 (s), 912 (m), 846 (w) cm -1 . 1 H NMR (CDCl 3 , 300 MHz): δ 1.47 (9 H, s), 1.64 (1 H, br s, NH), 2.26 (2 H, qt, J 7.0, 1.5), 2.67 (2 H, t, J 7.0), 3.30 (2 H, s), 5.05 (1 H, dq, J 17.0, 1.5), 5.11 (1 H, dq, J 17.0, 1.5), 5.80 (1 H, ddt, J 17.0, 10.0, 7.0). 13 C NMR (CDCl 3 , 75.5 MHz): δ 28.0, 34.3, 48.3, 51.5, 80.9, 116.3, 136.1, 171.5. MS m/z (ES + ) 130, 184, 186 (MH + ), 240, 244, 300, 322. HRMS m/z (ES + ) 186.1499 (MH + C 10 H 20 NO 2 requires 186.1494). 25 Acetic anhydride (1.50 equiv, 36.0 mmol, 3.40 mL) was added dropwise, at 0 °C, to a solution of crude secondary amine 10 (1.00 equiv, assumed 23.9 mmol, 4.43 g) in pyridine (25 mL). The cold bath was removed. The mixture was stirred at 20 °C for 1 h, then poured in 1 M HCl aqueous solution (0.10 L). The pH was adjusted to 1 by adding concentrated HCl aqueous solution. The mixture was extracted with Et 2 O (3 × 50 mL). The combined organic phases were washed with brine, then dried over MgSO 4 , filtered and concentrated to afford a pale yellow oil (4.43 g). Purification by flash column chromatography on silica gel, (EtOAc/heptane, 40%) yielded pure tert-butyl 2-(N-(but-3-en-1-yl)acetamido)acetate 11 (3.52 g, 15.5 mmol, 37% over two steps from tert-butyl chloroacetate). -12 -1.5), 5.12 (1 H, dq, J 17.0, 1.0), 5.78 (1 H, ddt, J 17.0, 10.0, 7.0). Minor rotamer: δ 1.48 (9 H, s), 2.03 (3 H, s), 2.28 (2 H, tdt, J 7.5, 7.0, 1.5), 3.44 (2 H, t, J 7.5), 3.90 (2 H, s), 5.03 (1 H, dq, J 10.0, 1.5), 5.07 (1 H, dq, J 17.0, 1.5), 5.78 (1 H, ddt, J 17.0, 10.0, 7.0). 13 C NMR (CDCl 3 , 75.5 MHz), 65 : 35 mixture of two rotamers: δ 21. 1, 21.5, 27.9, 28.0, 32.0, 32.9, 46.8, 48.2, 49.3, 51.7, 81.6, 82.4, 116.6, 117.6, 134.2, 135.2, 168.3, 168.4, 170.7, 170.8

Synthesis of 5b

Butyric anhydride (1.21 equiv, 24.0 mmol, 3.93 mL) was added dropwise, at 20 °C, to a solution of crude secondary amine 10 (prepared as described above for the synthesis of 5a, 1.00 equiv, assumed 19.9 mmol, 3.68 g) in pyridine (20 mL)

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

-13 -17.0), 5.76 (1 H, ddt, J 17.0, 10.0, 7.0). 13 C NMR (CDCl 3 , 75.5 MHz), 67 : 33 mixture of two rotamers: δ 13.9, 13.9, 18.4, 18.6, 28.0, 28.0, 32.1, 33.2, 34.7, 35.0, 47.0, 48.4, 48.5, 51.0, 81.5, 82.4, 116.5, 117.5, 134.3, 135.4, 168.5, 168.6, 173.1, 173.2

Typical procedures for the cycloaddition reactions under standard thermal conditions

Reaction of 5a with phenyl vinyl sulfone

A solution of 5a (1.00 equiv, 1.70 mmol, 360 mg) and phenyl vinyl sulfone (2.00 equiv, 3.41 mmol, 573 mg) in PhCl (17 mL) was stirred at reflux for 100 minutes. After cooling, the mixture was concentrated under reduced pressure to give the crude product (orange oil, 988 mg). Analysis by (2 H, m with the aspect of a dd, J 8.0, 7.0), 2.00 (1 H, ddd, J 13.5, 12.0, 5.5), 2.10 (1 H, ddd, J 13.5, 12.0, 4.0), 2.11 (1 H, ddd, J 13.5, 9.5, 7.5), 2.52 (1 H, ddd, J 10.0, 8.0, 6.5), 2.62 (1 H, ddd, J 13.5, 7.5, 7.0), 2.96 (1 H, ddd, J 10.0, 8.0, 7.0), 3.13 (1 H, ddd, J 14.0, 12.0, 4.0), 3.43 (1 H, ddd, J 14.0,

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

-14 -12.0, 5.5), 3.46 (1 H, dd, J 9.5, 7.0), 3.90 (1 H, t, J 7.5), 7.57 (2 H, br t, J 7.5), 7.59 (2 H, br t, J 7.5), 7.66 (1 H, br t, J 7.5), 7.69 (1 H, br t, J 7.5), 7.81 (2 H, br d, J 7.5), 8.02 (2 H, br d, J 7.5). 13 C NMR (CDCl 3 , 75.5 MHz): δ 18.4, 21.6, 28.0, 30.5, 32.7, 36.8, 44.5, 45.3, 53.2, 62.0, 65.5, 79.7, 81.9, 127.9, 128.4, 129.3, 129.4, 133.5, 133.6, 139.0, 141.2, 170.5.

Reaction of 5a with N-phenylmaleimide

A solution of 5a (1.00 equiv, 1.13 mmol, 238 mg) and N-phenylmaleimide (2.00 equiv, 2.26 mmol, 391 mg) in PhCl (10 mL) was stirred at reflux for 80 minutes. After cooling, the mixture was concentrated under reduced pressure to give the crude product (orange oil, 704 mg). Analysis by , 21.1, 28.1, 34.4, 37.3, 45.6, 51.2, 53.8, 63.6, 75.7, 82.2, 126.4, 128.6, 129.1, 131.9, 168.6, 175.5, 175.5 4, 23.2, 28.1, 33.3, 36.2, 44.9, 46.1, 47.5, 50.3, 55.4, 64.9, 82.7, 83.2, 126.2, 126.6, 128.7, 128.9, 129.2, 129.4, 131.9, 131.9, 167.6, 175.0, 175.5, 177.6, 178

Typical procedures for the cycloaddition reactions under microwave conditions

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Reaction of 5a with diethyl fumarate

p-Toluenesulfonic acid monohydrate (2% equiv, 20.0 µmol, 3.8 mg) was added to a solution of 5a (1.00 equiv, 1.00 mmol, 211 mg) and diethyl fumarate (1.00 equiv, 1.00 mmol, 164 µL) in PhCl (1.0 mL). The mixture was heated using a microwave apparatus (power: 250 W, temperature: 140 °C, ramp time: 2 min, hold time: 10 min, max. pressure: 10 bars). After cooling, saturated aqueous NaHCO 3 solution (20 mL) was added and the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product (378 mg). Purification by flash column chromatography on silica gel (EtOAc/heptane, gradient from 20 to 30%) afforded pure 19c (41. (1 H, dddd, J 12.0, 11.5, 11.5, 6.5), 1.48 (9 H, s), 1.83 (1 H, ddd, J 12.0, 6.0, 5.0), 1.96 (1 H, dqd, J 11.5, 7.0, 6.0), 2.59 (1 H, ddd, J 11.5, 8.5, 5.0), 2.86 (1 H, d, J 12.0), 2.95 (1 H, dd, J 8.5, 6.5), 3.63 (1 H, dd, J 12.0, 11.5), 3.93 (1 H, d, J 11.5), 4.03-4.29 (4 H, m). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14. 1, 14.2, 14.5, 25.4, 28.0, 33.3, 39.2, 47.6, 49.0, 58.8, 60.8, 60.9, 66.6, 73.0, 82.0, 168.2, 171.1, 172 (1 H, dddd, J 12.5, 9.0, 7.5, 5.0), 1.95 (1 H, dddd, J 12.5, 8.5, 8.0, 6.0), 2.18 (1 H, dqd, J 9.0, 7.5, 6.0), 2.86 (1 H, ddd, J 10.0, 8.5, 7.5), 2.92 (1 H, d, J 10.5), 3.09 (1 H, ddd, J 10.0, 8.0, 5.0), 3.60 (1 H, t, J 10.5), 4.02 (1 H, d, J 10.5), 4.02-4.26 (4 H, m). 13 C NMR (CDCl 3 , 75.5 MHz): δ 13.9, 14. 1, 14.2, 28.0, 30.8, 31.7, 42.8, 48.6, 50.3, 56.5, 60.8, 60.9, 67.3, 77.2, 82.1, 168.5, 172.6, 172.6. MS m/z (ES + ) 236,282,326,328,329,330,350,384 (1 H, dddd, J 12.5, 12.0, 10.5, 8.5), 2.08 (1 H, dddd, J 12.5, 7.0, 6.5, 1.0), 2.28 (1 H, dqd, J 12.0, 7.0, 6.5), 2.70 (1 H, ddd, J 10.5, 9.5, 7.0), 2.95 (1 H, ddd, J 9.5, 8.5, 1.0), 3.53 (1 H, d, J 11.5), 3.96 (1 H, dd, J 11.5, 7.5), 3.98-4.26 (4 H, m), 4.03 (1 H, d, J 7.5). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14.0, 14. 1, 14.2, 20.6, 28.0, 36.3, 43.8, 46.3, 51.7, 53.8, 60.6, 60.9, 65.3, 73.0, 81.7, 170.7, 171.4, 171.8 8.0, 7.5, 6.5), 2.08 (1 H, qdd, J 7.0, 6.5, 5.5), 2.83-3.00 (2 H, m), 3.52 (1 H, d, J 11.0), 3.88 (1 H, dd, J 11.0, 8.0), 3.95-4.22 (2 H, m), 4.11 (1 H, d, J 8.0), 4.16 (2 H, q, J 7.0). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14.0, 14. 2, 16.1, 26.6, 28.0, 34.3, 42.1, 46.7, 48.6, 51.5, 60.5, 60.9, 64.8, 74.7, 81.8, 170.7, 171.5, 171.9 (1 H, dd, J 10.5, 9.0), 4.03-4.23 (2 H, m), 4.12 (2 H, q, J 7.0), 4.41 (1 H, d, J 10.5). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14.0, 14. 2, 14.2, 20.0, 28.0, 32.5, 45.3, 47.9, 48.5, 55.5, 60.5, 60.7, 66.0, 77.2, 81.7, 169.3, 171.2, 171.9 13 C NMR (CDCl 3 , 75.5 MHz): δ 14.0, 14. 1, 15.0, 22.7, 28.0, 31.5, 44.3, 47.0, 48.8, 49.1, 60.5, 60.7, 66.3, 77.3, 81.9, 169.5, 171.2, 171.7 4.27 (4 H, m). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14.0, 14. 1, 14.2, 14.6, 16.7, 27.9, 33.1, 39.1, 40.0, 47.4, 49.3, 53.8, 60.7, 60.8, 68.0, 75.1, 81.8, 168.2, 171.4, 172.3.

Reaction of 5b with diethyl maleate

p-Toluenesulfonic acid monohydrate (2% equiv, 20.0 µmol, 3.8 mg) was added to a solution of 5b (1.00 equiv, 1.00 mmol, 239 mg) and diethyl maleate (1.00 equiv, 1.00 mmol, 162 µL) in PhCl (1.0 mL). The mixture was heated using a microwave apparatus (power: 250 W, temperature: 140 °C, ramp time: 2 min, hold time: 10 min, max. pressure: 10 bars). After cooling, saturated aqueous NaHCO 3 solution (20 mL) was added and the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product (400 mg). Analysis by GC/MS and NMR spectroscopy showed full conversion of 5b and the production of 3-tert-butyl 1,2-diethyl 7-methyl-7a-propylhexahydro-1Hpyrrolizine-1,2,3-tricarboxylate, mainly as the diastereoisomers 22a and 22b (ratio 48 : 52). The diastereoisomers 21 were also observed, in minor amounts. Purification by flash column chromatography on silica gel (EtOAc/heptane 20%) afforded various mixtures of diastereoisomers of 3-tert-butyl 1,2-diethyl 7-methyl-7a-propylhexahydro-1H-pyrrolizine-1,2,3-tricarboxylate (total amount 331 mg, 804 µmol, 80%). After a second flash column chromatography, 22a was isolated [contaminated with a minor amount of 21a] (44.9 mg, 109 µmol, 11%), as well as pure 22b (36.5 mg, 88.6 µmol, 9%).

22a: Colourless oil. d, J 9.0), 3.43 (1 H, dd, J 10.5, 9.0), 4.00-4.21 (4 H, m), 4.25 (1 H, d, J 10.5). 13 C NMR (CDCl 3 , 75.5 MHz): δ 13.7,14.1,14.1,14.9,18.1,28.0,32.8,36.1,46.8,47.2,48.6,56.2,60.5,60.5,66.5,77.2,81.3,169.4,171.4,172.1. MS 1.26 (1 H, m), 1.40 (1 H, dddd, J 12.0, 11.0, 9.5, 8.0), 1.48 (9 H, s), 1.50 (1 H, m), 1.53-1.70 (2 H, m), 1.85 (1 H, dddd, J 12.0, 8.5, 7.0, 3.5), 2.33 (1 H, dquint, J 11.0, 7.0), 2.66 (1 H, ddd, J 11.0, 9.5, 3.5), 2.87 (1 H, ddd, J 11.0, 8.5, 8.0), 3.24 (1 H, d, J 11.0), 3.36 (1 H, dd, J 11.0, 10.5), 4.09 (2 H, q, J 7.0), 4.10 (2 H, q, J 7.0), 4.20 (1 H, d, J 10.5). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14. 1, 14.1, 14.7, 15.2, 16.9, 28.0, 31.9, 35.2, 39.0, 47.4, 48.6, 50.1, 60.4, 60.6, 66.7, 79.8, 81.4, 169.7, 171.4, 171.8 .0), 4.12 (2 H, q, J 7.0), 4.17 (2 H, AB part of an ABX 3 system, δ A 4.13, δ B 4.21, J AB 18.0, J AX 7.0, J BX 7.0). 13 C NMR (CDCl 3 , 75.5 MHz): δ 14. 1, 14.2, 24.7, 25.5, 26.8, 27.5, 46.5, 52.0, 52.3, 54.3, 58.6, 60.6, 60.9, 62.6, 69.1, 76.0, 170.8, 172.2, 172.8. 10 M HCl aqueous solution (about 0.1 mL) was added into a solution of a sample of 24 (1.00 equiv, 186 µmol, 71.8 mg) in EtOAc (1.0 mL). The mixture was concentrated under reduced pressure to give a viscous pale yellow oil (82.4 mg). This crude product had turned solid after a few days. Recrystallisation from EtOAc afforded pure 24.HCl as colourless crystals that were suitable for Xray diffraction (53.8 mg, 126 µmol, 69%). 13 C NMR (CDCl 3 , 100.6 MHz): δ 13.98, 14.04, 24.5, 25.2, 27.7, 27.8, 45.0, 52.1, 53.8, 58.9, 59.6, 62.0, 62.3, 71.8, 74.4, 77.2, 167.6, 168.1, 168.9.

24.HCl

Figure 1 .

 1 Figure 1. Typical HPLC/MS (ES + ) TIC chromatogram of the crude product obtained after the thermal reaction of aminocyclopropane 5a with a dipolarophile. The result shown here corresponds to the reaction with N-phenylmaleimide presented in Table 1, entry 8. Diastereoisomers of compounds 15 (M = 384 g / mol) and 16 (M = 557 g / mol) are clearly visible.

Figure 2 .

 2 Figure 2. Ortep view of 15a. Displacement ellipsoids are drawn at the 30% probability level.

Scheme 7 .

 7 Scheme 7. Cycloaddition of 23 with diethyl fumarate.

Figure 3 .

 3 Figure 3. Ortep view of 24.HCl. Displacement ellipsoids are drawn at the 30% probability level.

Figure 4 .

 4 Figure 4. Examples of pyrrolizidine alkaloids having an alkyl substituent at the ring junction carbon atom.

  Thermoquest Navigator (ES + ), LCT Micromass (low-and high-resolution ES + ), Hewlett-Packard Quad GC-MS engine spectrometer via direct injection (positive CI, NH 3 ) and JEOL GCmate II (high-resolution EI) spectrometers. Underlined m/z values indicate the base peaks. 4.2. Procedures for the preparation of the substrates 5

11 :

 11 Colourless oil. C 12 H 21 NO 3(227.30): calcd. C 63.41, H 9.31; found C 63.23, H 9.38. IR (neat): ν 3433 (br), 2979, 2931, 1742 (s), 1654 (s), 1428 (m), 1368 (m), 1225 (m), 1157 (s), 917 (w), 853 (w) cm -1 . 1 H NMR (CDCl 3 , 300 MHz), 65 : 35 mixture of two rotamers. Major rotamer: δ 1.46(9 H, s), 2.15 (3 H, s), 2.33 (2 H, tdt, J 7.5, 7.0, 1.5), 3.41 (2 H, t, J 7.5), 3.96 (2 H, s), 5.10 (1 H, dq, J 10.0,

22b:

  Colourless solid. M.p. 77.5-78°C. IR (neat): ν 2959, 2934, 2873 (w), 1745 (m), 1721 (s), 1365 (m), 1333 (m), 1205 (m), 1156 (s), 1141 (m), 1102 (m), 1069 (m), 1018 (m), 845 cm -1 . 1 H NMR (CDCl 3 , 300 MHz): δ 0.88 (3 H, t, J 7.0), 1.06 (3 H, d, J 7.0), 1.21 (3 H, t, J 7.0), 1.24 (3 H, t, J 7.0),

Table 1 .

 1 Ratios Estimated by HPLC/MS. PVS = phenyl vinyl sulfone; NPM = N-phenylmaleimide.b Two identical experiments were carried out. The ratios found were 54 : 46 and 50 : 50 respectively. The average value is displayed in the table.c Obtained as a mixture of diastereoisomers that were not separated. d Phenyl vinyl sulfone was introduced in portions of 0.1 equiv, at intervals of 10 minutes. e A mixture of both compounds was obtained. The diastereoisomers were not separated.

			of the 1,3-dipolar cycloaddition/conjugate addition-1,3-dipolar cycloaddition
	products, depending on several reaction parameters.
		dipola-
		rophile a	pTSA			ratio 13/14 (from PVS)
	Entry	(equiv)	(equiv)	method	time	or 15/16 (from NPM) a	Tentative yields
	1	PVS (2)	0	conventional reflux	4 h	24 : 76
	2	PVS (2)	10%	conventional reflux	2 h	19 : 81
	3	PVS (1)	0	conventional reflux	3 h	53 : 47
	4	PVS (1)	10%	conventional reflux	2 h	52 : 48 b	13 (15%) c 14 (34%) c
	5	PVS (1) c	0	conventional reflux	3 h	53 : 47
	6	PVS (1) d	10%	conventional reflux	2 h	40 : 60
	7	PVS (1)	0	microwave heating	10 minutes	56 : 44	13 + 14 (60%) e
	8	NPM (1)	10%	conventional reflux	1 h	60 : 40	15 (28%) c 16 (28%) c
	9	NPM (1)	0	microwave heating	5 minutes	73 : 27

a

  . MS (EI) m/z126, 130, 145, 154, 162, 186, 227 ([M] +• ). HRMS (EI) m/z 227.1519 (M +• C 12 H 21 NO 3 +• requires 227.1516). Cyclopentylmagnesium chloride (1.95 M in Et 2 O, 3.00 equiv, 12.7 mmol, 6.52 mL) was added dropwise, over 10 min, to a cooled (0 °C), stirred solution of amide 11 (1.00 equiv 4.24 mmol, 963 mg) and titanium(IV) isopropoxide (1.50 equiv, 6.35 mmol, 1.88 mL) in THF (50 mL). After 30 min of stirring at 0 °C, H 2 O (1.0 mL) was added, the cold bath was removed and the flask was exposed to air. Stirring was maintained until decolouration (1 h). The reaction mixture was filtered through a pad of celite (rinsing: Et 2 O, 2 × 50 mL). The combined organic solutions were dired over Na 2 SO 4 , filtered and concentrated under reduced pressure to give a yellow oil (834 mg). Purification by flash column chromatography on silica gel treated with a few drops of Et 3 N (EtOAc/heptane 25%) yielded pure tert-butyl 2-(1-methyl-2-azabicyclo[3.1.0]hexan-2-yl)acetate 5a (563 mg, 2.66 mmol, 63%).

		0), 3.32 (1 H, t, J
	8.0). 13 C NMR (CDCl 3 , 75.5 MHz): δ 7.9, 19.3, 22.2, 26.0, 28.0, 46.1, 50.3, 54.6, 80.5, 170.5. MS
	(positive CI, NH 3 ) m/z 110, 156, 210, 212 (MH + ), 213. MS (EI) m/z 110, 111, 155, 211 ([M] +• ).
	HRMS (EI) m/z 211.1577 (M +• C 12 H 21 NO 2	+• requires 211.1567).

Note : this reaction was repeated several times under identical conditions. Yields were in the range 54-63% (7 runs). 5a: Colourless oil. C 12 H 21 NO 2 (211.30): calcd. C 68.21, H 10.02; found C 68.39, H 9.91. IR (neat): ν 3409 (br), 2975, 2932, 2869, 2817 (w), 1747 (s), 1727 (m), 1453 (w), 1391 (w), 1367 (m), 1257 (w), 1223 (w), 1150 (s) cm -1 . 1 H NMR (CDCl 3 , 300 MHz): δ 0.09 (1 H, dd, J 8.0, 5.5), 0.73 (1 H, t, J 5.5), 1.15 (1 H, dt, J 8.0, 4.0), 1.34 (3 H, s), 1.47 (9 H, s), 1.74 (1 H, dd, J 11.0, 7.0), 1.93 (1 H, m), 2.03 (1 H, ddd, J 11.0, 7.0, 4.0), 3.16 (2 H, AB system, δ A 2.80, δ B 3.53, J AB 16.

  . MS m/z (ES + ) 222, 223, 254, 278 (MNa + ), 279. HRMS m/z (ES + ) 278.1744 (MNa + C 14 H 25 NNaO 3 requires 278.1732).

	Cyclopentylmagnesium chloride (1.99 M in Et 2 O, 3.00 equiv, 15.0 mmol, 7.54 mL) was added
	dropwise, at 0 °C, over 10 min, to a stirred solution of amide 12 (1.00 equiv, 5.00 mmol, 1.28 g) and
	titanium(IV) iso-propoxide (1.50 equiv, 7.50 mmol, 2.22 mL) in THF (0.10 L). After 30 min of
	stirring at 0 °C, H 2 O (5.0 mL) was added, the cold bath was removed and the flask was exposed to
	air. Stirring was maintained until decolouration (1 h). The reaction mixture was filtered through a
	pad of celite (rinsing: Et 2 O, 2 × 50 mL). The combined organic solutions were dried over Na 2 SO 4 ,
	filtered and concentrated under reduced pressure to give a yellow oil (1.80 g). Purification by flash
	column chromatography on silica gel (EtOAc/heptane, gradient from 10 to 20%) yielded pure tert-
	butyl 2-(1-propyl-2-azabicyclo[3.1.0]hexan-2-yl)acetate 5b (866 mg, 3.62 mmol, 72%).

Note : another run performed on 12.7 mmol scale gave 5b in 62% yield. 5b: Colourless oil. IR (neat): ν 2930, 2869, 1747 (m), 1726 (m), 1455 (w), 1366 (m), 1256 (w), 1223 (w), 1145 (s), 958, 917, 849 cm -1 . 1 H NMR (CDCl 3 , 300 MHz): δ 0.10 (1 H, dd, J 8.0, 5.5), 0.68

(1 H, dd, J 5.5, 4.5)

, 0.90 (3 H, t, J 7.0), 1.20

(1 H, ddd, J 8.5, 8.0, 4.5), 1.22-1.51

(3 H, m), 1.46 (9 H, s), 1.76 (1 H, m), 1.91 (1 H, m), 1.97 (1 H, m), 1.99 (1 H, m), 3.14 (2 H, AB system, δ A 2.80, δ B 3.47, J AB 16.0), 3.28 (1 H, m).

13

C NMR (CDCl 3 , 75.5 MHz): δ 6.

9, 14.3, 19.3, 20.8, 26.3, 28.1, 34.3, 49.8, 50.2, 54.8, 80.5, 170.8

. MS m/z (ES + ) 184, 185, 206, 262 (MNa + ). HRMS m/z (ES + ) 262.1789 (MNa + C 14 H 25 NNaO 2 requires 262.1783).

  1 H NMR spectroscopy revealed the presence of 15a as the major product. Purification by flash column chromatography on silica gel (EtOAc/heptane, gradient from 10% to 100%) afforded pure 15a (101 mg, 263 µmol, 23%). Several other purifications by flash column chromatography on silica gel (EtOAc/heptane, gradient from 10% to 100% or EtOAc/CH 2 Cl 2 , gradient from 0% to 30%) or by recrystallisation (EtOAc) yielded pure samples of several adducts of the reaction: 15a (total amount 116 mg, 302 µmol, 27%), 15b (total amount 21.8 mg, 56.6 µmol, 5%), 16b (total amount 8.1 mg, 14.5 µmol, 1%) and 16a (total amount 56 mg, 100 µmol, 9%). Colourless crystals.. C 22 H 28 N 2 O

	Major product 15a:

4 (384.48): calcd. C 68.73, H 7.34; found C 68.52, H 7.53. IR (neat)

: ν 2966 (m), 2933 (w), 2875 (w), 1779 (w), 1746 (m), 1711 (s, C=O), 1496 (m), 1377 (m), 1366 (m), 1184 (m), 1174 (m), 1164 (m), 1156 (m), 1147 (m), 1128 (m), 1106 (m), 757 (m) cm -1 . 1 H NMR (CDCl 3 , 400 MHz): δ 1.09 (3 H, d, J 7.0), 1.15 (3 H, s), 1.51 (9 H, s), 1.59 (1 H, dddd, J 13.0, 9.0, 8.5, 7.0), 2.12 (1 H, dddd, J 13.0, 8.5, 8.0, 4.0), 2.46

(1 H, ddq, J 8.5, 8.0, 7.0), 2.82 (1 H, ddd, J 10.0, 8.5, 7

.0), 3.07

(1 H, d, J 8.5)

, 3.10 (1 H, ddd, J 10.0, 9.0, 4.0), 3.83

(1 H, dd, J 8.5, 8.0), 4.

04

(1 H, d, J 8.0), 7.25 (2 H, br d, J 7.5), 7.37

(1 H, br t, J 7.0), 7.45 (2 H, br dd, J 7.5, 7.0).

13

C NMR (CDCl 3 , 100.6 MHz): δ 16.1

  4 mg, 108 µmol, 11%), a 60 : 40 mixture of 19c and 19d (14.8 mg, 38.5 µmol, 4%), pure 19d (21.6 mg, 56.3 µmol, 6%), a 50 : 50 mixture 19d and 19a (30.9 mg, 80.5 µmol, 8%), pure 19a (126 mg, 328 µmol, 32%), a 65 : 35 mixture of 19a and 19b (78.1 mg, 204 µmol, 20%) and pure 19b(20.5 mg, 53.4 µmol, 5%). These amounts correspond to a total of 333 mg of cycloaddition diastereoisomers 19a-d (869 µmol, 87%).

	Major product 19a: Colourless oil. IR (neat): ν 2975, 2934, 2876, 1726 (s), 1367 (m), 1280, 1231
	(m), 1147 (s), 1096, 1031 (m), 846 cm -1 . 1 H NMR (CDCl 3 , 300 MHz): δ 0.89 (3 H, d, J 7.0), 1.23 (3
	H, t, J 7.0), 1.25 (3 H, s), 1.28 (3 H, t, J 7.0), 1.45

  .1. MS m/z (EI)136, 208, 209, 236, 282, 310, 383 (M +• ). MS m/z (ES + ) 282, 306, 328, 329, 350, 384 (MH + ), 406 (MNa + ), 407. HRMS m/z (ES + ) 406.2216 (MNa + C 20 H 33 NNaO 6 requires 406.2206).

	19b: Colourless oil. IR (neat): ν 2975, 1726 (s), 1454, 1367, 1231, 1146, 1096, 1031, 846, 737 cm -1 .
	1 H NMR (CDCl 3 , 300 MHz): δ 0.92 (3 H, d, J 7.5), 1.24 (3 H, t, J 7.0), 1.26 (3 H, t, J 7.0), 1.41 (3 H,
	s, H6), 1.48 (9 H, s), 1.58

  (MH + ), 406 (MNa + ). HRMS m/z (ES + ) 406.2216 (MNa + C 20 H 33 NNaO 6 requires 406.2206).

	19c: Colourless oil. IR (neat): ν 2974, 1730 (s), 1367 (m), 1212 (m), 1150 (s), 1030 (m) cm -1 . 1 H
	NMR (CDCl 3 , 300 MHz): δ 0.87 (3 H, s), 1.02 (3 H, d, J 7.0), 1.23 (3 H, t, J 7.0), 1.27 (3 H, t, J 7.0),
	1.43 (9 H, s), 1.72

  . MS m/z (ES + )282, 306, 328, 329, 350, 406 (MNa + ), 578. HRMS m/z (ES + ) 406.2199 (MNa + C 20 H 33 NNaO 6 requires 406.2206). The mixture was heated using a microwave apparatus (power: 250 W, temperature: 140 °C, ramp time: 2 min, hold time: 10 min, max. pressure: 10 bars). After cooling, saturated aqueous NaHCO 3 solution (20 mL) was added and the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product (383 mg). Purification by flash column chromatography on silica gel (EtOAc/heptane 30%) afforded pure 19d (7.1 mg, 18.5 µmol, 2%), a 43 : 57 mixture of 19d and 19a(15.6 mg, 40.6 µmol, 4%), pure 19a(17.8 mg, 46.4 µmol, 5%), a 41 : 59 mixture of 19a and 20a (91.1 mg, 237 µmol, 24%), pure 20a (68.3 mg, 178 µmol, 18%), a 56 : 44 mixture of 20a and 20b (9.1 mg, 23.7 µmol, 2%) and pure 20b (49.9 mg, 130 µmol, 13%). These amounts correspond to a total of 259 mg of cycloaddition diastereoisomers 19 and 20 (675 µmol, 67%).

	4.4.2. Reaction of 5a with diethyl maleate
	p-Toluenesulfonic acid monohydrate (2% equiv, 20.0 µmol, 3.8 mg) was added to a solution of 5a
	(1.00 equiv, 1.00 mmol, 211 mg) and diethyl maleate (1.00 equiv, 1.00 mmol, 162 µL) in PhCl (1.0
	mL). Major product 20a: Colourless oil. IR (neat): ν 2975, 2933, 2874 (w), 1727 (s), 1367 (m), 1245,
	1207 (m), 1151 (s), 1097 (m), 1064, 1046, 1030, 1015, 846, 836 cm -1 . 1 H NMR (CDCl 3 , 300 MHz):
	δ 1.03 (3 H, d, J 6.5), 1.04 (3 H, s), 1.20 (3 H, t, J 7.0), 1.25 (3 H, t, J 7.0), 1.50 (9 H, s), 1.52 (1 H,
	m), 1.85 (1 H, m), 1.86 (1 H, m), 2.71 (1 H, ddd, J 11.0, 9.5, 5.5), 2.90 (1 H, m), 3.13 (1 H, d, J 9.0),
	3.53

  . MS m/z (ES + )282, 306, 328, 329, 350, 384 (MH + ), 406 (MNa + ). HRMS m/z (ES + ) 406.2208 (MNa + C 20 H 33 NNaO 6 requires 406.2206).NaHCO 3 solution (20 mL) was added and the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give the crude product (355 mg). Analysis by 1 H NMR spectroscopy revealed the presence of 3tert-butyl 1,2-diethyl 7-methyl-7a-propylhexahydro-1H-pyrrolizine-1,2,3-tricarboxylate, mainly as the diastereoisomer 21a. Purification by flash column chromatography on silica gel (

	ACCEPTED MANUSCRIPT
	EtOAc/heptane
	10%) afforded pure 21c (8.6 mg, 20.8 µmol, 2%), a 50 : 50 mixture of 21c and 21d (6.5 mg, 15.7
	µmol, 2%), pure 21d (26.9 mg, 65.3 µmol, 8%), a 33 : 67 mixture of 21d and 21a (28.1 mg, 68.2
	M µmol, 8%) and a 70 : 30 mixture of 21a and 21b (110 mg, 267 µmol, 32%). These amounts correspond to a total of 180 mg of cycloaddition diastereoisomers 21 (438 µmol, 52%). Major product 21a: 1 H NMR (CDCl 3 , 300 MHz): δ 0.89 (3 H, d, J 7.0), 0.95 (3 H, m), 1.23 (3 H, t, J 7.0), 1.27 (3 H, t, J 7.0), 1.36-1.64 (4 H, m), 1.46 (1 H, dddd, J 12.0, 11.5, 6.0, 4.5), 1.47 (9 H, s), 1.78 (1 H, ddd, J 12.0, 6.0, 5.5), 1.98 (1 H, qdd, J 7.0, 6.0, 5.0), 2.59 (1 H, ddd, J 11.5, 9.0, 5.5), 2.95 A N U S C R I P T (1 H, dd, J 9.0, 6.0), 3.13 (1 H, d, J 12.0), 3.59 (1 H, dd, J 12.0, 11.5), 3.81 (1 H, d, J 11.5), 4.03-
	A C C E P T E D
	4.4.3. Reaction of 5b with diethyl fumarate
	p-Toluenesulfonic acid monohydrate (4.0% equiv, 33.4 µmol, 6.4 mg) was added to a solution of 5b
	-17 -

(1.00 equiv, 835 µmol, 200 mg)

and diethyl fumarate (1.00 equiv, 835 µmol, 137 µL) in PhCl (1.0 mL). The mixture was heated using a microwave apparatus (power: 250 W, temperature: 140 °C, ramp time: 2 min, hold time: 10 min, max. pressure: 10 bars). After cooling, saturated aqueous

  H, m), 2.70 (1 H, ddd, J 10.0, 9.0, 6.0), 2.85 (1 H, ddd, J 10.0, 6.0, 2.0), 3.09(1 H,

	1 H NMR (CDCl 3 , 300 MHz): δ 0.83 (3 H, t, J 7.0), 1.09 (3 H, d, J 6.5), 1.20 (3
	H, t, J 7.0), 1.20-1.60 (2 H, m), 1.25 (3 H, t, J 7.0), 1.38 (2 H, m), 1.50 (9 H, s), 1.52 (1 H, m), 1.78
	(1 H, m), 1.82 (1

  m/z (ES + ) 334, 356, 357, 378, 412 (MH + ), 434 (MNa + ), 435. HRMS m/z (ES + ) 434.2509 (MNa + C 22 H 37 NNaO 6 requires 434.2519).

Cycloaddition of 23 with diethyl fumarate p

  . MS m/z (ES + )206, 334, 356, 357, 378, 412 (MH + ), 434 (MNa + ), 435. HRMS m/z (ES + ) 434.2505 (MNa + C 22 H 37 NNaO 6 requires 434.2519). -Toluenesulfonic acid monohydrate (5.0% equiv, 12.5 µmol, 2.4 mg) was added to a solution of 23 (1.00 equiv, 250 µmol, 53.3 mg) and diethyl fumarate (1.00 equiv, 250 µmol, 43.0 mg) in PhCl (0.25 mL). The mixture was heated using a microwave apparatus (power: 300 W, temperature: 180 °C, ramp time: 2 min, hold time: 10 min, max. pressure: 10 bars). After cooling, the reaction mixture was diluted with CH 2 Cl 2 (10 mL), washed with 1 M NaOH aqueous solution (2.0 mL) and H 2 O (5.0 mL), then dried over Na 2 SO 4 , filtered and concentrated to afford the crude product (162 mg). Purification by flash column chromatography on silica gel (EtOAc/heptane 20%) afforded pure 3,4-diethyl 2methyl 5-cyclopentyl-5-(methoxymethyl)pyrrolidine-2,3,4-tricarboxylate 24 (63.0 mg, 163 µmol, 65%). Thick pale yellow oil.1 H NMR (CDCl 3 , 300 MHz): δ 1.24 (3 H, t, J 7.0), 1.25 (3 H, t, J 7.0),1.28-1.88 (8 H, m), 2.26 (1 H, m), 2.39 (1 H, br s, NH), 3.19 (3 H, s), 3.30 (1 H, d, J 11.0), 3.32 (2 H, AB system, δ A 3.23, δ B 3.41, J AB 9.5), 3.50(1 H, dd, J 11.0, 10.0), 3.74 (3 H, s), 3.83 (1 H, d, J 10

	4.5.

24:

:

  Colourless crystals. M.p. 145.6-148.6 °C.1 H NMR (CDCl 3 , 400 MHz): δ 1.28 (3 H, t, J 7.0), 1.29 (3 H, t, J 7.0), 1.47-1.67(8 H, m), 2.64 (1 H, tt, J 10.0, 8.0), 3.34 (3 H, s), 3.59 (1 H, d, J 11.0), 3.77 (2 H, AB system, δ A 3.76, δ B 3.78, J AB 10.5), 3.85 (1 H, dd, J 11.0, 10.0), 3.88 (3 H, s), 4.20 (2 H, br q, J 7.0), 4.25 (2 H, AB part of an ABX 3 system, δ A 4.23, δ B 4.27, J AB 11.0, J AX 7.0, J BX 7.0), 4.75(1 H, d, J 10.0), 7.56 (1 H, br s, NH), 12.55 (1 H, br s, NH).

Acknowledgements

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

-19 -This work was funded by the Institut de Chimie des Substances Naturelles (ICSN), École Polytechnique and the CNRS. We are grateful to Dr Angèle Chiaroni (X-ray crystallography) for her valuable help. We also wish to thank Marie-Françoise Bricot (elemental analysis), Michel Levart (CI and EI MS analyses), as well as the members of the HPLC and mass spectroscopy services of the Institut de Chimie des Substances Naturelles for their contribution to this work. Y. S. also wishes to thank Prof. S. Z. Zard for his encouragement and constant support.

Supplementary data

Supplementary data related to this article can be found at http://xx.xx.


Joseph E Sanfilippo

Adam A Nguyen

Laurence Garczarek

Jonathan A Karty

Suman Pokhrel

Johann A Strnat

Frédéric Partensky

Wendy M Schluchter

David M Kehoe
email: dkehoe@indiana.edu

Interplay between differentially expressed enzymes contributes to light color acclimation in marine Synechococcus

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Introduction

Members of the cyanobacterial genus Synechococcus are distributed throughout the marine environment, constituting a global population of approximately 7 x 10 26 cells [START_REF] Flombaum | Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus[END_REF]. The ubiquity of this genus is partially due to the wide diversity of its photosynthetic pigments, which allows for niche occupancy of a large variety of light environments [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF][START_REF] Ong | An unusual phycoerythrin from a marine cyanobacterium[END_REF]. There are three main pigment types of Synechococcus strains adapted to distinct spectral niches in the marine environment: blue light specialists, green light specialists, and blue-green generalists [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF][START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF].

These three pigment types differ by the composition of their light harvesting complexes, known as phycobilisomes (PBS). PBS are typically fan-shaped, with a central core that is appressed to the photosynthetic or thylakoid membranes, and multiple rods which extend from the core and provide increased surface area for photon capture. PBS consist of phycobiliproteins, apoproteins containing one to three covalently attached chromophores that absorb specific wavelengths of light [START_REF] Glazer | Light guides. Directional energy transfer in a photosynthetic antenna[END_REF], and linker proteins, which have structural and energy transfer roles [START_REF] Ong | Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy-transfer pathways in Synechococcus-spp phycoerythrins[END_REF]. Although there are several kinds of phycobiliproteins, all are α/β heterodimers. In marine Synechococcus, rods always contain the phycobiliprotein phycocyanin and usually contain one or two different forms of phycoerythrin called phycoerythrin-I (PE-I) and phycoerythrin-II (PE-II) [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF][START_REF] Ong | An unusual phycoerythrin from a marine cyanobacterium[END_REF][START_REF] Ong | Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy-transfer pathways in Synechococcus-spp phycoerythrins[END_REF] ~495 nm] and green-light absorbing phycoerythrobilin (PEB) (Absmax ~550 nm) [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF][START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. The total PUB:PEB ratio in all of the rods of the PBS determines if a strain is a blue or green light specialist. Blue light specialists have a high PUB:PEB ratio, while green light specialists have a low PUB:PEB ratio. Blue-green generalists are capable of modifying their PUB:PEB ratio in response to the ambient blue-green ratio through a process known as type IV chromatic acclimation or CA4 [START_REF] Palenik | Chromatic adaptation in marine Synechococcus strains[END_REF][START_REF] Everroad | Biochemical bases of type IV chromatic adaptation in marine Synechococcus spp[END_REF]. In the CA-4 capable strain Synechococcus sp. RS9916, each CpeA/CpeB dimer contains four PEB and one PUB in green light, and three PEB and two PUB in blue light. Each MpeA/MpeB dimer has four PEB and two PUB in green light, and two PEB and four PUB in blue light [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF].

Enzymes known as phycobilin lyases attach light harvesting chromophores on phycobiliproteins to conserved cysteine residues, forming thioether bonds [START_REF] Fairchild | Phycocyanin Alpha-Subunit Phycocyanobilin Lyase[END_REF][START_REF] Scheer | Biliprotein maturation: the chromophore attachment[END_REF]. Most lyases have high specificity for both particular chromophores and individual cysteine residues [START_REF] Scheer | Biliprotein maturation: the chromophore attachment[END_REF][START_REF] Bretaudeau | CyanoLyase: a database of phycobilin lyase sequences, motifs and functions[END_REF][START_REF] Schluchter | Phycobiliprotein biosynthesis in cyanobacteria: structure and function of enzymes involved in post-translational modification[END_REF]. While much is known about lyases capable of attaching phycocyanobilin (Absmax ~660 nm) [START_REF] Fairchild | Phycocyanin Alpha-Subunit Phycocyanobilin Lyase[END_REF][START_REF] Fairchild | Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase[END_REF][START_REF] Shen | Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002[END_REF][START_REF] Shen | Biogenesis of phycobiliproteins: I. cpcS-I and cpcU mutants of the cyanobacterium Synechococcus sp. PCC 7002 define a heterodimeric phyococyanobilin lyase specific for beta-phycocyanin and allophycocyanin subunits[END_REF][START_REF] Saunee | Biogenesis of phycobiliproteins: II. CpcS-I and CpcU comprise the heterodimeric bilin lyase that attaches phycocyanobilin to CYS-82 OF beta-phycocyanin and CYS-81 of allophycocyanin subunits in Synechococcus sp. PCC 7002[END_REF][START_REF] Zhao | Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins[END_REF][START_REF] Zhao | Lyase activities of CpcS-and CpcT-like proteins from Nostoc PCC7120 and sequential reconstitution of binding sites of phycoerythrocyanin and phycocyanin beta-subunits[END_REF][START_REF] Zhou | The cpcE and cpcF genes of Synechococcus sp. PCC 7002. Construction and phenotypic characterization of interposon mutants[END_REF], less is known about the enzymes involved in attaching PEB [START_REF] Wiethaus | CpeS is a lyase specific for attachment of 3Z-PEB to Cys82 of {beta}-phycoerythrin from Prochlorococcus marinus MED4[END_REF][START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF] and PUB [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Blot | Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyaseisomerase[END_REF][START_REF] Mahmoud | Adaptation to blue light in marine Synechococcus requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases[END_REF]. Despite the rich chromophore diversity found in marine Synechococcus, only two phycobilin lyases, RpcG and MpeZ, have been characterized so far in this group [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Blot | Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyaseisomerase[END_REF]. RpcG attaches PUB to cysteine 83 (C83) of the phycocyanin α-subunit [START_REF] Blot | Phycourobilin in trichromatic phycocyanin from oceanic cyanobacteria is formed post-translationally by a phycoerythrobilin lyaseisomerase[END_REF] and MpeZ attaches PUB to MpeA-C83 [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF]. These are dual function enzymes that have both lyase and isomerase activity, the latter allowing them to convert PEB into PUB. MpeU is required for attachment of PUB [START_REF] Mahmoud | Adaptation to blue light in marine Synechococcus requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases[END_REF], but its precise function remains to be characterized. The attachment and isomerization of PEB to PUB to phycobiliproteins by lyase-isomerases is critical since these cyanobacteria have no known pathway that directly synthesizes PUB.

Blue-green generalists capable of CA4 comprise the most abundant pigment type, making up approximately 40% of the global Synechococcus population of the oceans [START_REF] Grebert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF]. By differentially producing PEB and PUB in response to changes in ambient light color, bluegreen generalist strains can optimize light harvesting in a variety of light color environments. Interestingly, CA4 has evolved twice, through acquisition of related, but distinct, genomic islands. CA4-A strains contain a genomic island encoding the PUB lyaseisomerase MpeZ, while CA4-B strains possess a genomic island encoding MpeW, an as yet uncharacterized member of the CpeY-MpeY-MpeZ family [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. Both CA4-A and CA4-B genomic islands also encode two master regulators, FciA and FciB. In the model CA4-A strain Synechococcus sp. RS9916, these proteins inversely control the expression of mpeZ and two other genes located in the same genomic island, which are more highly expressed in blue light than green light [START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF][START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF]. During CA4-A, MpeA-C83, MpeA-C140, and CpeA-C139 bind PUB in blue light and PEB in green light [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF]. Of these three changes, MpeZ catalyzes the attachment of PUB to MpeA-C83 in blue light. However, the lyase responsible for attaching PEB to MpeA-C83 in green light is unknown.

Here, we investigate the role of a previously uncharacterized, putative PEB lyase named MpeY in CA4. We demonstrate that in the model CA4-A strain Synechococcus sp. RS9916, MpeY attaches PEB to MpeA-C83 in green light. Additionally, we show that the expression ratio of mpeY and mpeZ mRNA determines if PEB or PUB is attached to MpeA-C83 in green and blue light. By analyzing environmental isolates from across the world's oceans, we provide evidence that interplay between MpeY and MpeZ is globally conserved and that strains encoding only MpeY or MpeZ do not acclimate. Thus, the selective use of MpeY and MpeZ for the attachment of different chromophores at a single site within a phycobiliprotein is an evolutionary innovation that contributes to the process of CA4-A in marine Synechococcus.

Results

MpeY controls attachment of PEB in green light

The Synechococcus sp. RS9916 genomic region implicated in MpeA and MpeB biosynthesis contains the mpeBA operon and two putative phycobilin lyases, MpeY and MpeU (Fig 1A) [START_REF] Six | Diversity and evolution of phycobilisomes in marine Synechococcus spp.: a comparative genomics study[END_REF][START_REF] Mahmoud | Adaptation to blue light in marine Synechococcus requires MpeU, an enzyme with similarity to phycoerythrobilin lyase isomerases[END_REF]. While MpeU is not closely related to MpeZ, MpeY and MpeZ sequences from this strain share 47% amino acid sequence identity overall (Fig. S1A),

showing that these proteins are paralogs. Based on the genomic context of the mpeY gene and the relatedness of its encoded protein to MpeZ, we hypothesized that MpeY has a chromophore attachment role for either MpeA or MpeB.

Fluorescence spectroscopy was used to determine the relative excitation at 495 and 550 nm, which provides an estimate of the ratio of PUB to PEB in an mpeY interruption mutant (Fig. S1B) and control cells (Fig. 1B andC). In blue light, the PUB:PEB ratio of the mpeY mutant was indistinguishable from control cells, but in green light, PEB fluorescence decreased, relative to PUB fluorescence, in mpeY mutant cells. Therefore, MpeY is likely required for PEB attachment in green light. The possibility of polar effects was tested by cloning the mpeY gene and its promoter into an autonomously replicating plasmid and transforming it into the mpeY mutant. In green light, this plasmid restored the normal PUB:PEB ratio to the mpeY mutant (Fig. S2A), demonstrating that mpeY is necessary for PEB attachment in green light.

Whole cell absorbance spectroscopy showed that in blue light, mpeY mutant cells were indistinguishable from control cells but that in green light, mpeY mutant cells had lower levels of both PUB and PEB absorbance relative to chlorophyll levels (Fig. 1D andE). The effect on PEB was greater than the effect on PUB. Growth measurements demonstrated that mpeY mutant and control cells grew at the same rate in blue light but that in green light, mpeY mutant cells grew more slowly (Fig. S2B). This may be due to less efficient MpeA and MpeB biosynthesis in green light, resulting in a decrease in the number of complete PBS per cell and thus the overall amount of PUB and PEB in mpeY mutant cells. Together, these results demonstrate that MpeY is involved in PEB attachment in green light.

MpeY attaches PEB to MpeA-C83

The function of MpeY was further examined by using HPLC to separate the phycoerythrin subunits MpeA, MpeB, CpeA, and CpeB from PBS that had been purified from mpeY mutant cells grown in blue or green light. UV-VIS absorption spectroscopy of these proteins revealed that MpeA isolated from blue-light-grown mpeY mutant and control cells had identical profiles, while the PUB:PEB absorbance ratio for MpeA was higher in the mpeY mutant than in control cells after growth in green light (Fig. 2A andB). In addition, the absorption profiles of MpeB, CpeA, and CpeB isolated from mpeY mutant cells were indistinguishable from control cells after growth in either blue or green light (Fig. S3A-F).

We therefore conclude that among the four PE subunits and two light color conditions, MpeY is specifically required for PEB attachment to MpeA in green light.

In Synechococcus sp. RS9916, chromophores are attached to MpeA at C75, C83, and C140.

To determine which of these residues were targeted by MpeY, we used tandem mass spectrometry combined with UV-VIS spectroscopy to examine peptides containing these residues from mpeY mutant and control cells (Fig. S4). Cells grown in blue light had PUB attached to all three residues from both mpeY mutant and control cells (Fig. S5). In addition, for cells grown in green light, PUB was attached to C75 (Fig. 2C andD) and PEB was attached to C140 (Fig. 2G andH) from both cell types. The only observable difference in chromophorylation was at MpeA-C83 from cells grown in green light, where PEB was attached in control cells (Fig. 2E) and PUB was present in mpeY mutant cells (Fig. 2F).

Collectively, these data show that out of the 11 cysteines that are chromophorylated in the four PE subunits [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF], MpeY is required specifically for PEB attachment to MpeA-C83, and that this attachment can only be detected in green light.

The lyase activity of recombinant MpeY was examined using a heterologous plasmid coexpression system in Escherichia coli. Three different hexahistidine-tagged (HT-) versions of MpeA were used as substrates: wild type, HT-MpeA; a mutant with C83 replaced by alanine (A), HT-MpeA(C83A); and a mutant with C75 and C140 each replaced with A, HT-MpeA(C75A, C140A). We also expressed NusA-tagged (Nus) MpeY along with enzymes required for PEB synthesis in E. coli. After coexpression and cell lysis, HT-MpeA and mutant derivatives were purified using Ni-NTA affinity chromatography and analyzed using SDS-PAGE (Fig. 2I). Zinc-enhanced fluorescence was used to show that MpeY is sufficient to covalently attach PEB to C83 of MpeA (Fig. 2I) and thus is a PEB lyase.

mpeY -suppressor mutations increase mpeZ expression

MpeY attaches PEB to MpeA-C83 in green but not blue light, indicating that it plays a role in the chromophore changes at that residue during CA4. The mechanism through which MpeY operates was further characterized by selecting for spontaneous suppressor mutants with higher growth rates than the mpeY mutant. Passaging of three independent mpeY mutant cultures for approximately 100 generations selected for apparent suppressor mutations in the CA4 genomic island (Fig. 3A) that increased growth rate relative to the mpeY mutant (Fig. S6A) yet maintained their initial interruptions of mpeY (Fig. S6B).

Fluorescence spectroscopy analysis of each putative suppressor mutant revealed that all had an increased PUB:PEB ratio during growth in white light, compared to the original mpeY mutant and control cells (Fig. 3B), and were indistinguishable from one another, suggesting they could be caused by similar mutations.

A similarly high PUB:PEB ratio had been previously observed for an interruption mutant of fciB in all light conditions examined (4) and the suppressor mutants grew at a rate that was very similar to the fciB mutant (Fig. S6A). In addition, all of the suppressor mutants had a higher level of PUB absorbance than the original mpeY mutant and were phenotypically indistinguishable from the fciB mutant (Fig. S6C). These data collectively suggested that the suppressor mutations interrupted the normal expression of fciB.

Sequencing revealed a unique fciB mutation site in each of the three suppressor mutants examined, while no fciB mutation was present in control cells that were also passaged for 100 generations (Fig. 3A). Two of the suppressor mutants contained frameshift mutations in the genomic region encoding the N-terminal portion of FciB. One was due to a 5' TC 3' insertion (Suppressor 1) and the other to a 5' TC 3' deletion (Suppressor 3) in a region containing six 5' TC 3' repeats. Suppressor 2 contained a glycine to arginine missense mutation at residue 251 in the C-terminal portion of FciB, which is predicted to encode a helix-turn-helix DNA binding motif [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. Fluorescence excitation spectroscopy showed that the original mpeY mutant phenotype was restored after transformation of each suppressor strain with fciA and fciB, which previously complemented an fciB interruption mutant (4) (Fig. S7A), demonstrating that these fciB mutations caused the suppressor phenotypes.

Based on previous results showing that mpeZ transcript levels were constitutively high in both blue and green light in a fciB mutant (4), we tested the mpeY -suppressor mutants for elevated mpeZ transcript levels during growth in white light. RNA-Seq data demonstrated that absolute mpeZ RNA abundance was much higher than mpeY RNA abundance in blue light but much lower in green light, and that neither was ever completely absent (Fig. S7B).

RNA blot analyses showed that mpeY mutant cells had low levels of mpeZ transcripts, while both mpeY -suppressor and fciB mutant cells had approximately 50-fold higher mpeZ transcript levels (Fig. 3C). Therefore, the mpeY -suppressor mutants appear to modify the mpeY mutant growth phenotype in white light by inactivating fciB, leading to the elevation of the expression of mpeZ. Because MpeZ is known to attach PUB to MpeA-C83 [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF], the phenotypes of these suppressor mutants strongly suggest that the relative expression of mpeY and mpeZ controls the attachment of PEB versus PUB.

Interplay between MpeZ and MpeY controls chromophorylation of the same cysteine during CA4

Our discovery that mpeY suppressor mutants increased mpeZ expression led us to hypothesize that the MpeZ:MpeY ratio, driven by the relative expression levels of mpeZ and mpeY, controls whether PUB or PEB is attached at MpeA-C83 during CA4. We tested this by examining existing RNA-Seq data from fciA and fciB interruption mutants. As previously shown ( 4), the former is locked in CA4 green light phenotype and always has PEB at MpeA-C83, while the latter is locked in the CA4 blue light phenotype and always has PUB at MpeA-C83 (Fig. 4A,top). In addition, the mpeZ:mpeY expression ratio differed from the control in the fciA and fciB mutants, being high in the fciB mutant and low in the fciA mutant in both blue and green light (Fig. 4A, bottom). Thus, the mpeZ:mpeY mRNA expression ratio is correlated with the type of chromophore attached to MpeA-C83, suggesting that this ratio has a major role in determining whether PUB or PEB is attached to MpeA-C83 during CA4. This was directly tested by introducing an autonomously replicating plasmid expressing mpeY from its native promoter into wild-type cells. The mpeY RNA levels in this transformed line were 8-fold higher in green light and 14-fold higher in blue light than in wild type cells transformed with the empty vector (Fig. S7 C andD). Fluorescence emission spectra obtained from green-light-grown cells showed that PUB and PEB fluorescence spectra for the mpeY overexpression strain was indistinguishable from the empty vector control strain. However, for cells grown in blue light the mpeY overexpression strain exhibited a higher PEB to PUB fluorescence ratio than the empty vector control strain (Fig. 4, B andC). The simplest and most reasonable explanation for this result is that overproduced MpeY was outperforming MpeZ in blue light, leading to the addition of PEB instead of PUB at MpeA-C83. Therefore, we conclude that interplay between MpeZ and MpeY controls which of these two chromophores are attached at this residue during CA4.

Interplay between MpeY and MpeZ is widespread, suggesting a paradigm for pigment diversification in the marine environment

Our data show that in Synechococcus sp. RS9916, a strain isolated from the Gulf of Aqaba in the Red Sea, MpeY and MpeZ both act on the same residue as part of CA4. To explore if interplay between MpeY and MpeZ is widespread, we examined 20 strains isolated from seven regions around the globe that encode MpeY and/or MpeZ (Table S1), analyzing the phylogenetic relatedness of these enzymes (Fig. 5, right). Nine of these isolates encoded only MpeY, one isolate encoded only MpeZ, and 10 isolates encoded both MpeY and MpeZ (Fig. 5, left). While the single isolate encoding only MpeZ, BIOS-E4-1, was a blue light specialist (PUB rich), all nine isolates that only encode MpeY were green light specialists (PEB rich). Nine of the 10 strains, isolated from six different oceanic regions, encoded both MpeY and MpeZ and were blue-green generalists capable of CA4. The single exception, MVIR-18-1, was previously found to lack significant blue-green regulation of its mpeZ gene [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. From an evolutionary point of view, the phylogenetic analysis of these two enzymes (Fig. 5, right) shows that they derived from a common lyase ancestor and that after duplication, these proteins diverged structurally and functionally in a way that maintained attachment site specificity while diverging in the type of chromophore that each attached. Taken together, these findings support the widespread ability of two paralogous enzymes, the lyase MpeY and the lyase-isomerase MpeZ, to act in a concerted fashion on the same residue, thereby contributing to the pigment diversity observed in marine Synechococcus.

Discussion

The successful expansion of Synechococcus throughout the lit zone of the world's oceans likely relied in part on the diversification of its photosynthetic light harvesting abilities.

Blue light specialists, green light specialists, and blue-green generalists have evolved to incorporate an optimal combination of chromophores in their PBS to efficiently utilize solar energy in all light color niches encountered in the underwater environment. Specific energy absorption and transfer characteristics of each type of PBS are determined by which chromophore is added at each cysteine attachment site throughout the light-harvesting structure. These additions are catalyzed by phycobilin lyases and lyase-isomerases, making this a pivotal group of enzymes for successful light color adaptation of Synechococcus in the marine environment. Marine Synechococcus strains have up to 14 genes per genome that are predicted to encode phycobilin lyases that attach chromophores to distinct cysteine residues within the apoproteins of the PBS, although it is yet to be determined how specificity is achieved between each lyase and its cognate residue [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Bretaudeau | CyanoLyase: a database of phycobilin lyase sequences, motifs and functions[END_REF][START_REF] Schluchter | Phycobiliprotein biosynthesis in cyanobacteria: structure and function of enzymes involved in post-translational modification[END_REF] The mpeY and mpeZ genes co-occur in many Synechococcus strains and, in most case, this results into the ability for these cells to perform CA4 (Fig. 5). The only isolate (MVIR-18-1) that has both mpeY and mpeZ but is not a blue-green specialist provides additional support of our model because it has been shown that there is little to no blue-green light regulation of mpeZ gene expression in MVIR-18-1, making this strain unable to carry out normal CA4 [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. The only strain (BIOS-E4-1) that has mpeZ but not mpeY is a blue light specialist and also does not differentially express its mpeZ gene [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. As recently suggested [START_REF] Grebert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF], BIOS-E4-1 is likely to have previously possessed both mpeZ and mpeY, then lost mpeY and the light color regulation of mpeZ, leading to a blue light specialist phenotype.

Interestingly, the BIOS-E4-1 genotype is similar to the mutants that arose in our mpeY - suppressor screen, where inactivating mpeY led to more rapidly growing mutants (Fig. S6) that lost CA4 by expressing mpeZ at blue light levels even in green light conditions (Fig. 3C). BIOS-E4-1 also lacks fciA and fciB. The loss of these CA4 regulators might have been the next evolutionary step after the inactivation of mpeY and the generation of a fciB suppressor mutant. The inactivation of fciB would have made FciA useless, leading to eventual loss of mpeY, fciB, and fciA from the genome of these mutants. Thus, our inactivation of mpeY may have initiated a series of genetic changes that parallel an evolutionary process that is occurring in the oceans. In the natural environment, such BIOS-E4-1-like genotypes seem to predominate in some permanently iron-poor areas such as the tropical South Pacific Ocean [START_REF] Grebert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF].

The evolution of a light-color-regulated process in which two enzymes vie for the attachment of two different chromophores to the same site is an innovative, efficient way to add flexibility to light harvesting capability in photosynthetic organisms. This system, which involved the duplication of the common ancestor of MpeY and MpeZ, the divergence of their expression and functions, but the retention of their site of action (Fig. 5), may serve as a paradigm for how phenotypic plasticity of pigmentation is widely generated with paralogous genes in marine Synechococcus. Because CA4 has evolved more than once and appears in approximately 40% of all Synechococcus cells in the ocean [START_REF] Grebert | Light color acclimation is a key process in the global ocean distribution of Synechococcus cyanobacteria[END_REF], the capacity to alter light harvesting clearly provides strains with a distinct evolutionary advantage.

The interplay between MpeY and MpeZ is an outstanding example of how horizontally acquired gene products interact with components encoded in the core genome to confer a novel trait, such as CA4. Although it has been previously established that horizontal acquisition of genomic islands can drive the evolution of novel traits in marine Synechococcus [START_REF] Paz-Yepes | Role of a microcin-C-like biosynthetic gene cluster in allelopathic interactions in marine Synechococcus[END_REF][START_REF] Stuart | Genomic island genes in a coastal marine Synechococcus strain confer enhanced tolerance to copper and oxidative stress[END_REF][START_REF] Dufresne | Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria[END_REF], precisely how these events influence the subsequent phenotype of the organism is still not well understood [START_REF] Dorman | H-NS-like nucleoid-associated proteins, mobile genetic elements and horizontal gene transfer in bacteria[END_REF].

In summary, we have discovered that interplay between the lyase MpeY and lyaseisomerase MpeZ controls the attachment of two different light harvesting chromophores, each absorbing a different wavelength, to the same residue of a light harvesting protein in marine Synechococcus. Despite their evolutionary relatedness, MpeY attaches PEB and MpeZ attaches PUB, and in strains containing both mpeY and mpeZ, the mpeY: mpeZ expression ratio determines if PEB or PUB is attached. Additionally, isolates with only mpeY or mpeZ are incapable of CA4. We conclude that the interplay between MpeY and MpeZ is important for CA4 in marine Synechococcus and hypothesize that other forms of this interplay occur between other paralogous lyase/lyase-isomerase pairs in this important group. In addition to furthering our basic understanding of a globally important marine picophytoplankton, the previously unidentified ability of two enzymes to vie for attachment of spectrally different chromophores to the same cysteine residue will provide new tools for further development of multi-color fluorescence labeling technology.

Materials and methods

Strains and growth conditions. Growth conditions were as previously described [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF].

Details are provided in Supporting Information. Wild type cells were genetically unaltered Synechococcus sp. RS9916 cells [START_REF] Fuller | Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea[END_REF] and control cells were wild type cells containing a mini-Tn5 transposon insertion [START_REF] Mccarren | Transposon mutagenesis in a marine Synechococcus strain: Isolation of swimming motility mutants[END_REF]. This insertion did not affect CA4 and provided comparable kanamycin resistance to the mpeY interruption mutant used in the study. A Biotek Synergy-MX spectrofluorometer was used to generate fluorescence spectra as described [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF].

In vivo heterologous expression and purification of recombinant proteins. Plasmid constructs are provided in Supporting Information. Recombinant proteins were expressed and purified from E. coli BL21 (DE3) as previously described [START_REF] Shen | Identification and characterization of a new class of bilin lyase: the cpcT gene encodes a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the beta-subunit of phycocyanin in Synechococcus sp. PCC 7002[END_REF][START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF]. Details are provided in Supporting Information. Cell lysis, centrifugation and protein purification were conducted as previously described [START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF].

Protein analysis by spectroscopy and gel-electrophoresis. Most polypeptide samples

were resolved by polyacrylamide gel electrophoresis (PAGE, 15% w/v) in the presence of sodium dodecyl sulfate (SDS) and visualized as previously described [START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF]. mpeY disruption/complementation/overexpression. Transformation of Synechococcus sp. RS9916 by conjugation was performed as previously described [START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF][START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Brahamsha | A genetic manipulation system for oceanic cyanobacteria of the genus Synechococcus[END_REF]. Details are provided in Supporting Information.

HPLC and MS-MS analysis of phycobiliproteins. Phycobilisomes were purified as described [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Collier | Chlorosis induced by nutrient deprivation in Synechococcus sp. strain PCC 7942: not all bleaching is the same[END_REF]. HPLC was used to separate each phycobiliprotein subunit and samples were digested with trypsin as described previously [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF][START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF]. HPLC separated and trypsindigested phycobiliprotein subunits were analyzed by UV-VIS and MS/MS as described [START_REF] Shukla | Phycoerythrin-specific bilin lyase-isomerase controls bluegreen chromatic acclimation in marine Synechococcus[END_REF].

Suppressor analyses. To select for mpeY mutant suppressors, three independent mpeY mutant cell lines were grown in white light for approximately 100 generations. Details are provided in Supporting Information.

RNA analyses. RNA sequencing data were further analyzed from previous experiments (4), except that instead of values for the blue light control being set to 100 and values normalized across each replicate separately, the raw values were averaged. RNA blots were performed as previously described [START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF][START_REF] Seib | A turquoise mutant genetically separates expression of genes encoding phycoerythrin and its associated linker peptides[END_REF]. Values were normalized as previously described [START_REF] Sanfilippo | Self-regulating genomic island encoding tandem regulators confers chromatic acclimation to marine Synechococcus[END_REF][START_REF] Seib | A turquoise mutant genetically separates expression of genes encoding phycoerythrin and its associated linker peptides[END_REF]. Details are provided in Supporting Information.

Comparative genomics and phylogenetic analyses. Data on mpeY and mpeZ gene content were retrieved from the Synechococcus genomes described in Table S1 using the Cyanorak v2 database (www.sb-roscoff.fr/cyanorak). Phylogenetic reconstructions were performed using Maximum likelihood (ML; PhyML v3.0), Neighbor Joining (NJ, Phylip 3.69), and Bayesian inference (BI; MrBayes v3.1.2) as previously described [START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]. The ML tree with all bootstrap or posterior probability values from the different methods was then manually refined using Archaeopteryx Version 0.9901 [START_REF] Han | phyloXML: XML for evolutionary biology and comparative genomics[END_REF]. Maximum Likelihood (ML) analysis of MpeY and MpeZ using the distantly related phycobilin lyase CpeY as a root [START_REF] Biswas | Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481[END_REF]. Colored circles indicate the pigment phenotype (see insert). Series of three numbers at nodes correspond to bootstrap values for ML and Neighbor-Joining analyses and Bayesian posterior probabilities (PP, ranging between 0 and 1). Only values higher than 60% for bootstrap values and 0.60 for PP are shown on the phylogenetic tree. Underline denotes Synechococcus sp. RS9916, the strain used throughout this study. S2.

Suppressor analyses. Cultures were plated and individual colonies were picked and tested by PCR amplification to confirm interruption of mpeY. PCR amplification and sequencing were used to identify spontaneous mutations in fciB. pJS3 was transformed into suppressor mutant lines to complement the fciB mutations. Generation time was calculated as the log2 of the change in whole cell absorbance at 750 nm over time during the linear range of growth.

RNA Analyses. Cells were acclimated to blue or green light for at least 7 days, RNA was purified, and libraries were generated using an Epicentre (Illumina) ScriptSeq complete bacteria kit. "Normalized reads" for mpeZ and mpeY were calculated using custom Perl scripts. For RNA blots, 10 μg of RNA was added per lane. Blots were probed with DNA probes to mpeY or mpeZ generated by PCR amplification and radiolabeling, using primers mpeY-probe-for and -rev and mpeZ-probe-for and -rev (shown in Table S2).

directions of priming for PCR amplification reactions. S3 contains the exact retention times, m/z ratios, and visible absorption maxima for each peptide. Spec R , contains autonomously replicating vector WT pJS9 Spec R , contains vector expressing mpeY mpeY - Kan R , plasmid insertion disrupting mpeY mpeY -pJS1 Kan R , Spec R , contains autonomously replicating vector mpeY -pJS9 Kan R , Spec R , contains vector expressing mpeY mpeY -S1 Kan R , TC deletion in 5' end of fciB mpeY -S2 Kan R , G251R missense mutation in fciB mpeY -S3

Kan R , TC insertion in 5' end of fciB mpeY -S1 pJS1 Kan R , Spec R , contains autonomously replicating vector mpeY -S2 pJS1 Kan R , Spec R , contains autonomously replicating vector mpeY -S3 pJS1 Kan R , Spec R , contains autonomously replicating vector mpeY -S1 pJS3 Kan R , Spec R , contains vector expressing fciA and fciB mpeY -S2 pJS3 Kan R , Spec R , contains vector expressing fciA and fciB mpeY -S3 pJS3 Kan R , Spec

  The a and b subunits of PE-I are encoded by the cpeBA operon and are called CpeA and CpeB, while the a and b subunits of PE-II are encoded by the mpeBA operon and are called MpeA and MpeB. These heterodimers are located in different regions of the rods, with CpeA and CpeB in the coreproximal regions and MpeA and MpeB in the core distal regions. Phycoerythrin in marine Synechococcus strains contain two different types of chromophores, blue-light absorbing phycourobilin (PUB) [maximum absorbance (Absmax)

  . Our finding that MpeY and MpeZ attach different chromophores to the same residue increases our understanding of lyase residue specificity and provides additional insights into how different chromophores are attached. The MpeY overexpression experiment, which resulted in an increase of PEB:PUB ratio (Fig 4B), strongly suggests that the relative proportion of the MpeY and MpeZ enzymes in 9916 cells determines which chromophore, PEB or PUB, is bound to MpeA-C83. This site binds PUB in green-light grown mpeY mutant (Fig 2F), demonstrating that MpeZ is present under these conditions. Therefore, in wild-type cells grown in green light, even though the mpeZ transcript level is 6-times lower than in blue light (Shukla et al. 2012), MpeZ must be present, but outcompeted by MpeY.

Fig 1 .

 1 Fig 1. MpeY controls PEB attachment in green light. (A) mpeY is located in the PE-II

Fig 2 .

 2 Fig 2. MpeY attaches PEB to MpeA-C83. Absorbance spectra of purified MpeA protein

Fig 3 .

 3 Fig 3. mpeY-suppressor mutations increase mpeZ expression. (A) mpeY -suppressor

Fig 4 .

 4 Fig 4. The ratio of transcripts encoding MpeY and MpeZ determines the

Fig 5 .

 5 Fig 5. Interplay between MpeY and MpeZ is conserved throughout Synechococcus isolates

Fig 1 .

 1 Fig 1. MpeY controls PEB attachment in green light. (A) mpeY is located in the PE II

Fig 2 .Fig 3 .

 23 Fig 2. MpeY attaches PEB to MpeA-C83. Absorbance spectra of purified MpeA protein

Fig 4 .

 4 Fig 4. The ratio of transcripts encoding MpeY and MpeZ determines the

Fig 5 .

 5 Fig 5. Interplay between MpeY and MpeZ is conserved throughout Synechococcus

Fig S2 .

 S2 Fig S2. Complementation and growth rate analysis of the mpeY mutant. (A) Fluorescence

Fig S3 .

 S3 Fig S3. MpeY does not affect MpeB, CpeA, or CpeB chromophorylation. Absorbance

Fig S4 .

 S4 Fig S4. HPLC-MS data from tryptic digests of MpeA indicating the chromophore

Fig S5 .

 S5 Fig S5. MpeY does not affect MpeA chromophorylation in blue light. Absorbance

Fig

  Fig S6. mpeY -suppressor mutant analyses. (A) Generation time for mpeY mutant, mpeY -

Table S1 .

 S1 Description of strains used in Figure5This strain does perform CA4 but the amplitude of variation of the PUB:PEB is lower than in other CA4-A strains[START_REF] Humily | A gene island with two possible configurations is involved in chromatic acclimation in marine Synechococcus[END_REF]

	Strain_Name RCC* Subcluster Clade	Pigment_Type	Sea or Ocean	Region	References
	KORDI-49	-	5.1	WPC1 green-light specialist North Pacific Ocean	East China Sea	(26)
	M16.1	791	5.1	II	green-light specialist North Atlantic Ocean	Gulf of Mexico	(26)
	NOUM97013	2433	5.1	VII	green-light specialist South Pacific Ocean	-	(26)
	ROS8604	2380	5.1	I	green-light specialist North Atlantic Ocean	English Channel	(26)
	RS9907	2382	5.1	II	green-light specialist Red Sea	Gulf of Aqaba	(26)
	SYN20	2035	5.1	I	green-light specialist North Sea	-	(26)
	TAK9802	2528	5.1	II	green-light specialist South Pacific Ocean	Takapoto atoll	(2)
	WH7803	28	5.1	V	green-light specialist North Atlantic Ocean	Sargasso Sea	(2)
	MVIR-18-1	2385	5.1	I	green-light specialist North Sea	-	(7)
	WH8016	2535	5.1	I	CA4-A	North Atlantic Ocean	Woods Hole	(7)
	BIOS-U3-1	2533	5.1	CRD1	CA4-A	South Pacific Ocean	Chile upwelling	(7)
	BL107	515	5.1	IV	CA4-A	Mediterranean Sea	Spanish coast	(7)
	CC9311	1086	5.1	I	CA4-A	North Pacific Ocean	California Current	(7)
	CC9902	2673	5.1	IV	CA4-A	North Pacific Ocean	California Current	(7)
							Equatorial Pacific
	MITS9220	2571	5.1	CRD1	CA4-A	Pacific Ocean	Ocean	(7)
	PROS-9-1	328	5.1	Ib	CA4-A	Mediterranean Sea	-	(7)
	RS9916	555	5.1	IX	CA4-A	Red Sea	Gulf of Aqaba	(7)
	WH8020	2437	5.1	I	CA4-A	North Atlantic Ocean	-	(7)
	RCC307	307	5.3		CA4-A**	Mediterranean Sea	-	(7)
	BIOS-E4-1	2534	5.1	CRD1	blue-light specialist	South Pacific Ocean	South East Pacific	(7)
	*RCC, Roscoff Culture Collection Number
	**

Table S2 .

 S2 List of strains, plasmids, and primers used

	Strain	Description
	WT	Synechococcus sp. RS9916, isolated from the Red Sea
	Control	Kan R , mini-Tn5 insertion in uncharacterized gene RS9916_32112
	WT pJS1

Table S3 .

 S3 List of retention times, m/z ratios, and visible absorption maxima that indicate the bilins attached to cysteines 75, 83, and 140 in MpeA under various light conditions and genotypes. The letters in the graph column refer to the data presented in FigureS4.

	Graph	Light Color	Genotype	Peptide Sequence	Retention Time (min)	Ion A m/z Ion B m/z	Ion C m/z	VIS Peak, nm	Chromo -phore
	A	Green	WT		KC*75AT EGK	17.66 a	441.545 3+ 661.821 2+ 1322.637 1+	490	PUB
	B	Green	mpeY-	KC*75AT EGK	36.63 b	441.539 3+ 661.813 2+	n/a	490	PUB
	C	Green	WT		C*83KR	19.84 a	496.750 2+ 992.509 1+	n/a	550	PEB
	D	Green	mpeY-	C*83KR	29.54 b	331.498 3+ 496.748 2+	n/a	490	PUB
	E	Green	WT		NDGC*14 0SPR	25.24 a	667.786 2+ 445.526 3+ 1334.583 1+	550	PEB
	F	Green	mpeY-	NDGC*14 0SPR	49.22 b	445.519 2+ 667.778 2+	n/a	550	PEB
	Plasmid G	Blue	WT	Description KC*75AT EGK	17.71 a	441.546 3+ 661.818 2+ 1322.634 1+	490	PUB
	pMUT100 pRL528 H Blue I Blue	Kan R suicide vector used for homologous recombination mpeY-KC*75AT 25.99 c 395.688 4+ 527.252 3+ 790.384 2+ EGKEK Helper plasmid, carries mob WT C*83KR 14.34 a 496.749 2+ 992.499 1+ n/a	490 490	PUB PUB
	pRK24 pJASmpeY J Blue pJS1 K Blue	Conjugal plasmid, RK2 derivative mpeY-C*83KR 21.71 c 331.493 3+ 496.744 2+ pMUT100 derivative, for disruption of mpeY Spec R , autonomously replicating in RS9916 WT NDGC*14 0SPR 18.49 a 667.786 2+ 445.522 3+ 1334.603 1+ 992.506 1+	490 490	PUB PUB
	pJS3 pJS9 L	Blue	pJS1 derivative, for expression of fciA and fciB pJS1 derivative, for expression of mpeY mpeY--NDGC*14 0SPR 31.06 c 445.516 3+ 667.781 2+ 1334.588 1+	490	PUB
	pMpeA	Notes:		pCOLA Duet; produces 9916 HT-MpeA
	pMpeZ a. 60 minute LC gradient	pCDF Duet; produces 9916 NT-MpeZ
	pMpeA:C83A b. 120 minute LC gradient	pCOLADuet; produces 9916 HT-MpeA(C83A)
	pMpeA:C75,140A c. 105 minute LC gradient	pCOLADuet; produces 9916 HT-MpeA(C75,140A)
	pMpeY			pET(44b); produces 9916 Nus-MpeY
	pPcyA			pACYCDuet-1; produces Ho1 and HT-PcyA
	pPebS			pACYCDuet-1; produces Ho1 and NT-PebS
	Primer					Sequence (5' to 3')
	Mut-BamHI-mpeY-for		TGCTGGAAGCACTGGGGAG
	Mut-BamHI-mpeY-rev		ATCGGATCCCAACGTTCTTCAGCCCAACAACTGC
	mpeZ-probe-for				TTTTGGGCTGCACCGATACT
	mpeZ-probe-rev

R , contains vector expressing fciA and fciB ACGATGGCTCAGATTTCGCT mpeY-probe-for GCCAGAAGCTTCTTTGTGCACTCC

Acknowledgments

We thank members of the Kehoe laboratory, especially Lisa Wiltbank, as well as Andrian Gutu, Jeff Palmer, and Jake McKinlay for helpful discussions and comments. This research was supported by National Institutes of Health Training Grant to J.E.S. (T32-GM007757), National Science Foundation Grants to D.M.K. (MCB-1029414 and MCB-1818187) and to W.M.S. (MCB-1244339), and by the Office of the Vice Provost for Research at Indiana University, Bloomington, through its Bridge Funding Program to D.M.K. and by the French "Agence Nationale de la Recherche" Programs SAMOSA (ANR-13-ADAP-0010) for F.P. and L.G.

Supporting Information

Supporting Methods

Strains and Growth Conditions. Semi-continuous cultures were grown in polycarbonate culture flasks at 22°C in PCR-S11 media in constant light at an irradiance of 10 μmol photons m -2 s -1 from fluorescent white light bulbs unless noted (Chroma 75 T12; General Electric). Filters (LE716 Mikkel Blue and LE738 Jas Green; LEE Filters) were used to generate blue and green light. When necessary, 50 μg mL -1 kanamycin and/or 30 μg mL -1 spectinomycin was added to cultures. Absorbance spectra were produced using a Beckman DU640B spectrophotometer.

Plasmid construction. The plasmids and primers used are listed in Table S2. pJASmpeY was made by PCR amplification and cloning of a 750 bp internal region of mpeY into the BamHI site of pMUT100. pJS9 was made by PCR amplification and cloning of the promoter and ribosome-binding site of mpeY into the BamHI and AseI sites of pJS1. For the creation of expression constructs used in this study, each gene was amplified by PCR amplification from Synechococcus sp. RS9916 DNA using the primers listed in Table S2.

All amplified DNA and cloning junctions were checked by sequencing.

In vivo heterologous expression