Sunchu Prabhakar

Thomas Vivès

Vincent Ferrières

Thierry Benvegnu

Laurent Legentil
email: laurent.legentil@ensc-rennes.fr

Loïc Lemiègre
email: loic.lemiegre@ensc-rennes.fr

Fully enzymatic esterification/transesterification sequence for the preparation of symmetrical and unsymmetrical trehalose diacyl conjugates

Monoacyl and diacyl trehalose were synthesized in two steps from trehalose and carboxylic acids. The carboxylic acids were converted first into the corresponding 2,2,2-trifluoroethyl esters through a biocatalyzed esterification. The acyl donor was then transferred to the disaccharide through biocatalyzed transesterification. Thanks to microwave reaction conditions the transesterification proceeded selectively to the monoacyl trehalose or to the diacyl counterparts depending on the sole amount of acyl donor. These reaction conditions applied also for the preparation of unsymmetrical diacyl trehalose in a fully enzymatic sequence.

Introduction

Biocatalysis is nowadays part of the organic chemist toolbox for the design of synthetic schemes. It offers several advantages such as mild reaction conditions, selectivity, reduction of protection/deprotection steps and limited uses of toxic reagents or solvents. Indeed, the development of efficient biocatalysis processes dramatically enhances organic synthetic schemes. Among enzymatic processes, lipase mediated esterification is one of the most studied with the aim of reducing environmental impact of such chemical transformation that classically required acyl chlorides or toxic coupling agents. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] Lipases are used for numerous purposes such as kinetic resolution of asymmetric compounds, 2-6 hydrolysis [START_REF] Chen | [END_REF] or alcoholysis 8,9 of triglycerides, modification of saccharides [10][11][12][13][14][15][16][17][18] and polysaccharides 19 . The last two examples are of importance as they lead to various types of biologically active glycolipids. Indeed lipases are usually selective for the primary alcohol of hexosides which is useful for a selective protection with an acyl group [15][16][17] or for the selective functionalization of the C6 position with various acyl chains [10][11][12] . However, enzymatic esterifications often lead to moderate yields due to the equilibrium with the hydrolysis pathway. To circumvent this drawback, one would prefer transesterification of vinyl or alkyl esters in low water content media. This transesterification is much more efficient displacing the equilibrium towards the formation of the desired ester, the release of aldehydes or less nucleophilic alcohols being the driving force of the reaction.

Although the conversion yield is higher, this strategy definitely introduces a chemical step for the preparation of the required starting vinyl or alkyl esters.

We describe hereafter a fully enzymatic esterification/transesterification sequence applied to the preparation of glycolipids. It includes the preparation of the acylating agent and its reaction on an alcohol function both with the same enzymatic strategy. We developed this strategy on trehalose which is an important disaccharide involved for instance in the cell wall of mycobacteria. 20,21 Indeed, this sugar has two primary hydroxyl groups and the corresponding R e v i s e d m a n u s c r i p t mono and diesters are active compounds against this microorganism. [21][START_REF] Sakamoto | [END_REF][23][24] Besides, trehalose lipids have gained increased interest as biosurfactants for their potential applications in bioremediation technologies and oil/petroleum industry, and in biomedical/healthcare industry as antimicrobial and therapeutic agents. 25 Until now the preparation of such glycolipids requires multi-step synthesis [26][27][28][29] and lipase mediated esterifications are not efficient enough, conducing only to monoester derivatives 30 or to diesters compounds but in a long reaction time 31 . The synthetic scheme described here, includes two consecutive lipase-mediated steps starting from fatty acid and trehalose. A 2,2,2-trifluoroethyl ester would be prepared by an enzymatic esterification and this ester would be involved in an enzymatic transesterification step leading to the final glycolipid (Scheme 1). Trifluoroethyl esters have several advantages in terms of reactivity (good leaving group) and of access from trifluoroethanol. The challenge is i) to develop an efficient enzymatic access to 2,2,2-trifluoroethyl esters, and ii) to control the transesterification process towards the selective preparation of diacyl and/or monoacyl trehalose opening the access to unsymmetrical derivatives.

R e v i s e d m a n u s c r i p t

Scheme 1. Fully enzymatic strategy towards the selective esterification of trehalose.

Results and discussion

Lipase mediated transesterifications are dramatically improved when the starting ester is derived from vinyl or 2,2,2-trifluoroethyl moieties for instance. Among them we have chosen to work on 2,2,2-trifluoroethyl esters due to the availability 2,2,2-trifluoroethanol and their common use as acyl donor in enzymatic 13,[32][33][34] and non-enzymatic 35 transesterification reactions. These esters are classically prepared by reaction of 2,2,2-trifluoroethanol on activated carboxylic acid derivative such as acyl chloride or anhydride, or using Steglich esterification conditions (DCC/DMAP). 13,[36][37][38][39] No biocatalyzed methodology has been described so far. The development of a lipase mediated access to 2,2,2-trifluoroethyl esters would therefore result in a fully enzymatic two-step esterification process. We first optimized reaction conditions on linoleic acid by screening the type of lipase, solvent, type of activation (thermal or microwave) and amount of 2,2,2-trifluoroethanol (Table 1). To limit as much as possible the hydrolysis of a similar yield (72%), however reduction to 2 equivalents found the limit of the reaction and led to a poorer yield of 35% (Entries 8-9). Microwave activation is known to accelerate lipase mediated hydrolysis, esterification or transesterification. [START_REF] Izquierdo | [END_REF]4,[START_REF] Chen | [END_REF][40][41][42][43][44][45] We thus switched from thermal to microwave activation (180 W) but keeping the same reaction temperature (46 °C).

We were pleased to observe complete reactions after 1h instead of 12 h and in addition to increased yields (Entries 10-12). Indeed, whatever the solvent CAL-B (Imb) mediated esterifications were more efficient (Entries 10-12). As for the thermal activation, a maximum yield was reached in t-BuOH (82%, entry 12). It is noteworthy that similar yields were obtained under thermal or microwave conditions, the later impacted more the reaction time than the reaction yield. Again, attempts to reduce to 2 equivalents of CF 3 CH 2 OH gave a lower yield (42%, entry 13), so that 5 equivalents were optimal for this reaction. Having the activated esters in hands, we further studied the biocatalyzed monoacylation and diacylation of trehalose. This sugar is a a,a-1,1-disacharide of D-glucose. Its structure reveals two primary hydroxyl functions that are the less sterically hindered and the more nucleophilic sites. 46 Depending on the number of equivalent of fatty ester or on the reaction conditions one can expect mono (3) and diacylation (4) of trehalose (Table 2).

Based on the esterification procedure described above, reactions were carried out with the same lipase (CAL-B (Imb)) in t-BuOH, in the presence of activated molecular sieves and at 51 °C under thermal and microwave activation with linoleic acid or the corresponding 2,2,2trifluoroethyl linoleate as acylating agent. As previously observed, 30,31 the direct acylation of trehalose with 1.1 or 4 equivalents ( (Scheme 3). The branched esters (2d-e) provided the diacyl trehalose (4d-e) in lower yields ranging from 33 to 38%. As for the esterification step described above, the lipase have a reduced activity with such branched esters due to unfavorable steric hindrance within the active site affording lower yields compared to linear fatty chains. Interestingly, ester 2f bearing 2 cyclopronane moieties is a good model of mycolic acid conjugates. This ester was prepared from the linoleate 2a after adaptation of a known procedure. 47,48 Scheme 3. Enzymatic diacylation of trehalose Since the reaction conditions developed here can yield selectively mono trehalose conjugates we subsequently evaluated the ability of this approach for the synthesis of unsymmetrical diester derivatives (Scheme 4). This type of trehalose glycolipids, such as maradolipid, are found in several organisms and require multistep synthesis or the use of coupling agents (TBTU). 29,49 As a proof of concept, we developed the synthesis of the unsymmetrical diacyl trehalose 5 following our fully enzymatic sequence without any protection/deprotection steps.

As mentioned here before ( substrates on the initial rate of the reaction was evaluated using the Michaelis-Menten model (See supporting information). First the initial reaction rates (V0) increased with increasing concentration of either the acid or the alcohol. It demonstrated that no inhibition occurred by both substrates of the lipase or by the ester formed. The double reciprocal plot or Lineweaver-Burk plot 1/V0 versus the reciprocal of linoleic acid concentration at fixed trifluoroethanol concentration showed a set of parallel lines characteristic of a Ping-pong bi-bi mechanism. Such mechanism has been generally found with soluble or immobilized lipase. The resulting calculation for Km of both substrates gave the value of 0.7 mol.L -1 for the linoleic acid and 0.9 mol.L -1 for the trifluoroethanol. It means that both substrates bind to the enzyme with the same affinity. This low affinity is linked to the nature of the lipase, and its interfacial mechanism.

They are conforming to the ones found in the literature. [50][START_REF] Chowdhury | [END_REF][52]

Conclusion

The fully enzymatic strategy developed herein allowed the straightforward and efficient synthesis of trehalose glycolipid derivatives which represent important class of biologically active molecules. The final diacyl trehalose were obtained by a lipase mediated transesterification with 2,2,2-trifluoroethyl fatty esters. The choice for this later was particularly appropriate as it made possible the development of an enzymatic approach to the corresponding 2,2,2-trifluoroethyl fatty esters so that a unique biocatalyst was used for a two-step esterification/transesterification reaction. Thus, this work renews the access to symmetrical and also unsymmetrical diacyl trehalose compounds in an environmentally friendlier manner avoiding any classical esterification step.

Experimental

R e v i s e d m a n u s c r i p t 4.1 General methods

All reactions were carried out in oven-dried glassware. Optical rotations were measured at 20 °C on a Perkin-Elmer 341 polarimeter. NMR spectra were recorded on a Bruker Avance III 400 spectrometer operating at 400.13 MHz for 1 H and 100.61 MHz for 13 C. Chemical shifts are given in δ units (ppm) and referenced to CD 3 OD or CDCl 3 . Coupling constants J were calculated in Hertz (Hz). MS spectra were recorded on a Waters Micromass Q-TOF equipped with a Z-spray ion source.

General procedure for preparation of 2,2,2-trifluoroethyl fatty esters. Carboxylic acid

(1 equiv., typical quantity 100 mg), 2,2,2-trifluoroethanol (5 equiv.) and lipase (Immobilized CAL B, 93 mg per mmol of carboxylic acid, 3000 U/g) were placed in t-BuOH in the presence of activated molecular sieves (4Å) and under nitrogen atmosphere. The reaction mixture was placed in a microwave oven using a temperature control mode (180 W, 46 °C) for 1h. The supported lipase was removed by filtration and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (Cyclohexane/EtOAc) to afford the required compound.

2,2,2-trifluoroethyl linoleate 2a

The general procedure for the preparation of 2,2,2-trifluoroethyl esters has been applied to the synthesis compound 2a. The titled compound was isolated as a colorless oil in 82% yield. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] H NMR (400 MHz, CDCl 3 ) d (ppm) 5.45-5.27 (4H, m), 4.46 (2H, q, J H-F = 8.5 Hz), 2.84-2.68 (2H, m), 2.41 (2H, t, J = 7.5 Hz), 2.10-1.94 (4H, m), 1.66 (2H, m), 1.42-1.21 (14H, m), 0.88 (3H, t, J = 6.9 Hz); 13 C NMR (100 MHz, CDCl 3 ) d (ppm) 172. 28, 130.38, 130.14, 128.23, 128.03, 123.17 (q, J C-F = 277.1 Hz), 60.27 (q, J C-F = 36.6 Hz), 33.79, 31.68, 29.71, 29.50, 29.23, 29.20, 29.08, 27.36, 27.32, 25.78, 24.83, 22.73, 14.22; HRMS m/z calcd for C20H33O2F3Na

R e v i s e d m a n u s c r i p t

[M+Na] + : 385.2325, found: 385.2329.

2,2,2-trifluoroethyl oleate 2b

The general procedure for the preparation of 2,2,2-trifluoroethyl esters has been applied to the synthesis compound 2b. The titled compound was isolated as a yellowish oil in 80% yield. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] H NMR (400 MHz, CDCl 3 ) d (ppm) 5.44-5.28 (2H, m), 4.45 (2H, q, J H-F = 8.5 Hz), 2.40 (2H, t, J = 7.5 Hz), 2.10-1.92 (4H, m), 1.71-1.59 (2H, m), 1.41-1.18 (20H, m), 0.87 (3H, t, J = 6.6 Hz); 13

2,2,2-trifluoroethyl erucate 2c

The general procedure for the preparation of 2,2,2-trifluoroethyl esters has been applied to the synthesis compound 2c. The titled compound was isolated as a colorless oil in 75% yield. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] [M+Na] + : 443.3107, found: 443.3109.

2,2,2-trifluoroethyl 2-ethylhexanoate 2d

The general procedure for the preparation of 2,2,2-trifluoroethyl esters has been applied to the synthesis compound 2d. The titled compound was isolated as a yellowish oil in 68% yield. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] H R e v i s e d m a n u s c r i p t NMR (400 MHz, CDCl 3 ) d (ppm) 4.47 (2H, q, J H-F = 8.5 Hz), 2.42-2.33 (1H, m), 1.71-1.38 (4H, m), 1.37-1.17 (4H, m), 0.89 (3 H, t, J = 7.5 Hz), 0.87 (3 H, t, J = 7.3 Hz); 13 C NMR (100 MHz, CDCl 3 ) d (ppm) 174.85, 123.20 (q, J C-F = 278.1 Hz), 59.98 (q, J C-F = 36.4 Hz), 47.09, 31.68, 29.56, 27.06, 25.44, 22.67, 13.94, 11.71; HRMS m/z calcd for C10H17O2F3Na [M+Na] + : 249.1073, found: 249.1075.

2,2,2-trifluoroethyl 2-hexyldecanoate 2e

The general procedure for the preparation of 2,2,2-trifluoroethyl esters has been applied to the synthesis compound 2e. The titled compound was isolated as a colorless oil in 59% yield. [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] H NMR (400 MHz, CDCl 3 ) d (ppm) 4.46 (2H, q, J H-F = 8.5 Hz), 2.51-2.36 (1H, m), 1.69-1.54 (2H, m), 1.55-1.38 (2H, m), 1.35-1.15 (20H, m), 0.89 (6 H, t, J = 6.6 Hz); 13 C NMR (100 MHz, CDCl 3 ) d (ppm) 174.99, 123.22 (q, J C-F = 277.2 Hz), 59.98 (q, J C-F = 36.5 Hz), 45 Under nitrogen atmosphere a solution of 2a (0.20 g, 0.55 mmol) in dry CH 2 Cl 2 (2 mL) was cooled down to -23 o C, and diethyl zinc (1.0 M solution in hexane, 3.3 mL, 3.3 mmol) was added. The reaction mixture was stirred at the same temperature for 5 min before the dropwise addition of diiodomethane (0.5 mL, 5.5 mmol). After 12 h at -23 o C to 0 o C, the reaction mixture was treated with aqueous NH 4 Cl and extracted with Et 2 O. The combined organic phases were washed with saturated aqueous NaHCO 3 and brine and were dried over MgSO 4 . The solvent were removed under reduced pressure and the residue was purified by flash chromatography on silica gel (Cyclohexane/EtOAc) to afford ester 2f (0.2 g, 78%). [START_REF] Otera | Esterification: Methods, Reactions, and Applications[END_REF] NMR (100 MHz, CDCl 3 ) d (ppm) 172.22, 123.17 (q, J C-F = 277.1 Hz), 60.23 (q, J C-F = 36.6 Hz), 33.77, 32.06, 30.26, 30.05, 29.55, 29.38, 29.14, 29.05, 28.88, 28.19, 28.04, 24.85, 22.88, 16.20, 16.07, 16.02, 15.81, 15.75, 14.22, 11.16, 10.98; HRMS m/z calcd for C22H37O2F3Na

[M+Na] + : 413.2638, found: 413.2642.

General procedure for the acylation of trehalose.

General procedure. Trehalose (1 equiv., typical quantity 30 mg), acyl donor and lipase (immobilized CAL B, 126 mg per mmol of trehalose, 3000 U/g) were placed in t-BuOH under nitrogen atmosphere and in the presence of activated molecular sieves (4 Å, 10% w/w). The reaction mixture was placed in a microwave oven under temperature control (180 W, 51 o C) for 1 h. The supported lipase was removed by filtration and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel (CH 2 Cl 2 /MeOH) to afford required compounds.

6-linoleoyl trehalose 3a

The general procedure has been applied to the synthesis of monoester 3a. The titled compound was isolated as acolorless oil in 73% yield. [ The general procedure has been applied to the synthesis of diester 5 from 3a. The titled compound was isolated as a yellowish oil in 78% yield.

R e v i s e d m a n u s c r i p t

  s c r i p t a) The reaction was carried out in the presence of 5 equiv. of CF 3 CH 2 OH In the presence of 1.1 equiv. of 2,2,2-trifluoroethyl linoleate 2a, our reaction conditions led to the monoacyl trehalose 3a in 73% yield after 1h under microwave activation (Entry 4). It confirmed that the transesterification of trehalose occurred selectively towards the formation of the sole monoacylated trehalose 3a. In the presence of 4 equiv. of 2,2,2-trifluoroethyl linoleate 2a, the transesterification proceeded smoothly towards the diacyl derivative both under thermal (Entry 5) and microwave (Entry 6) activations. Again the reaction was much more efficient under microwave (180 W) leading to the diacyl trehalose 4a in 85% yield (Entry 6).

  s c r i p t Therefore, transesterification conditions were established for the selective mono-or diacylation of trehalose in the case of linoleate derivatives. The diacylation conditions were successfully applied to other fatty acid 2,2,2-trifluoroethyl esters (2) such as oleic (2b) or dicyclopropaneted stearic (2f) leading to the corresponding diacyl trehalose 4 in good yields (4a-b,f, 82-91%)

  (Imb), t-BuOH or n-hexane, Mw 180W, MS 4Å, 51°C, 1h

Scheme 4 .

 4 Scheme 4. Access to unsymmetrical diacyl trehalose by stepwise enzymatic acylations.

  H NMR (400 MHz, CDCl 3 ) d (ppm) 4.45 (2H, q, J H-F = 8.5 Hz), 2.40 (2H, t, J = 7.5 Hz), 1.71-1.61 (2H, m), 1.54-1.22 (18H, R e v i s e d m a n u s c r i p t m), 1.21-0.98 (2H, m), 0.89 (3H, t, J = 7.1 Hz), 0.86-0.53 (6H, m), (-0.22)-(-0.34) (2H, m); 13 C

  [α] 20 D +6.7 (c 1.0; MeOH); 1 H NMR (400 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 5.07-4.91 (6H, m), 4.73 (2H, d, J = 3.7 Hz), 3.99 (2H, dd, J = 12.0, 2.3 Hz), 3.90 (2H, dd, J = 12.0, 5.1 Hz), 3.64 (2H, ddd, J = 10.2, 5.

  s c r i p t

  the newly formed ester, all reactions were carried out in the presence of molecular sieves (MS 4 Å) that trapped the released water. The results of this optimization are gathered in Table1.

	Four lipases were selected: immobilized Candida antartica Lipases A and B (CAL-A (Imb)
	and CAL-B (Imb)), Candida rugosa Lipase (CRL) and porcine pancreas lipase (PPL). Under
	thermal activation (D, 46 °C), among these four lipases, CAL-B afforded the best yields after
	12h of reaction in t-BuOH (74%) and in the presence of 5 equivalents of CF 3 CH 2 OH (Entries
	1-4). Thus, we continued the study with CAL-B (Imb). Changing the solvent to toluene led to
	a very poor yield (<5%, entry 5) while THF and n-hexane provided lower yields (respectively
	65% and 52%, entries 6-7). Indeed, t-BuOH represents a good compromise in terms of
	hydrophilicity and hydrophobicity. The use of 10 equivalents of CF 3 CH 2 OH instead of 5 gave

Table 1 .

 1 Optimization of the enzymatic esterification of linoleic acid into 2,2,2-

trifluoroethyl linoleate Entry Lipase CF 3 CH 2 OH (equiv.)

	O	Lipase, CF 3 CH 2 OH, MS 4Å	O
	Solvent t-BuOH t-BuOH t-BuOH t-BuOH toluene THF n-hexane 12 h, D, 46 °C Conditions 12 h, D, 46 °C 12 h, D, 46 °C 12 h, D, 46 °C 12 h, D, 46 °C 12 h, D, 46 °C 12 h, D, 46 °C t-BuOH 12 h, D, 46 °C t-BuOH 12 h, D, 46 °C THF 1 h, Mw 180 W, 46 °C n-hexane 1 h, Mw 180 W, 46 °C t-BuOH 1 h, Mw 180 W, 46 °C t-BuOH 1 h, Mw 180 W, 46 °C Therefore, optimized conditions were settled as follow: CAL-B (Imb) lipase in the presence of Yield (%) 1 CAL-A 5 10 2 PPL 5 20 3 LCR 5 10 4 CAL-B 5 74 5 CAL-B 5 <5 6 CAL-B 5 65 7 CAL-B 5 52 8 CAL-B 10 72 9 CAL-B 2 35 10 CAL-B 5 73 11 CAL-B 5 59 12 CAL-B 5 82 13 CAL-B 2 42 7 solvent, conditions O 7 CF 3 1a 2a R e v i s e d 5 equivalents of CF 3 CH OH m a n u s c r i p t

2 OH, molecular sieve (4 Å) in t-BuOH and under a microwave activation of 1 h (180 W, 46 °C) (Scheme 2). These conditions were applied to other linear fatty acids 1 (oleic and erucic acids) with similar yields (2a-c, 75-82%). Surprisingly, the esterifications of branched fatty acids 1d-e were performed in a n-hexane/t-BuOH : 1:1 mixture in order to reach good conversions (2d-e, 59-68%). Concerning these two branched carboxylic acids we suppose that they do not fit perfectly within the catalytic site of the enzyme explaining the slightly lower yields. Scheme 2. Enzymatic esterification of fatty acids into 2,2,2-trifluoroethyl esters.

Table 2

 2 the presence of 4 equiv. of acid 1a, only the monolinoleate 3a was isolated in 42% yield.Moreover, with 1.1 equivalents, only traces of monoacyl derivative 3a were obtained.We therefore envisaged an in situ activation of the fatty acid by addition of CF 3 CH 2 OH into the reaction media following our optimized protocol. Interestingly, 4 equivalents of linoleic acid 1a in the presence of 5 equivalents of CF 3 CH 2 OH gave selectively after 2 h of reaction the monoacyl derivative 3a in 65% yield and only traces of diacyl compound 4a (Entry 3). It is noteworthy that this in situ procedure gave both better yield and selectivity compared to the reaction carried out without CF 3 CH 2 OH (Mono 42%, Di 15% entry 2). It revealed that the acylation of trehalose proceeded at least partly through a transesterification of the in situ formed 2,2,2-trifluoroethyl ester. Therefore, this transesterification is responsible for the high selectivity towards a monoacylation. Thus, we envisaged the stepwise esterification.

	O		CAL-B, CF 3 CH 2 OH 5 equiv.	O
	R	OH	t-BuOH or n-hexane, MS 4Å	R	O	CF 3
	1		microwave 180 W, 46°C, 1h	2
			O
			7	O	CF 3		O
	2,2,2-trifluoroethyl linoleate 2a, 82%		O	CF 3
			O		2,2,2-trifluoroethyl 2-ethylhexanoate 2d, 68%
			7	O	CF 3		O
	2,2,2-trifluoroethyl oleate 2b, 80%		O	CF 3
			O		2,2,2-trifluoroethyl 2-hexyldecanoate 2e, 59%
			11	O	CF 3
	2,2,2-trifluoroethyl erucate 2c, 75%

, Entries 1-2) of carboxylic acid 1a (R = H) were inefficient or required long reaction time. Almost no diacyl trehalose 4a was formed even in

Table 2 .

 2 Enzymatic esterification of linoleic acid and trehalose

			Yield (%)
	Entry Equiv. Conditions
			Mono 3a Di 4a	trehalose
	1	1.1	Microwave, 2 h traces	-	90
	2	4	Microwave, 2 h 42	15	35

Table 3 .

 3 Enzymatic transesterification of linoleic acid 2,2,2-trifluoroethyl ester by trehalose

				Yield (%)
	Entry Equiv. Conditions
				Mono 3a Di 4a	trehalose
	1	1.1	Microwave, 1 h 73	traces	<5
	2	4	Thermal, 18h	15	62	20
	3	4	Microwave, 1 h <5	85	<5

Table 2

 2

	, entry 4), the reaction of a first 2,2,2-trifluoroethyl linoleate
	(1.1 equiv.) gave the monoacyl trehalose 3a (Scheme 4, 73%). Starting from this isolated
	monolinoleyl trehalose 3a, the second acylation was performed with 3 equivalents of 2,2,2-
	trifluoroethyloleate 2b leading to the desired unsymmetrical diacyl trehalose 5 in 78% yield.

  C NMR (100 MHz, CDCl 3 ) d (ppm) 172.21, 130.16, 129.81, 123.16 (q, J C-F = 277.1 Hz), 60.24 (q, J C-F = 36.5 Hz), 33.75, 32.07, 29.92, 29.80, 29.68, 29.48, 29.23, 29.19, 29.09, 27.36, 27.35, 27.28, 24.84, 22.83, 14.21; HRMS m/z calcd for C20H35O2F3Na [M+Na] + : 387.2481,

found: 387.2480.

  Hz);13 C NMR (100 MHz, CDCl 3 ) d (ppm) 172.16, 130.52, 130.48, 123.19 (q, J C-F = 277.1 Hz), 60.22 (q, J C-F = 36.6 Hz), 33.75, 32.78, 32.11, 32.07, 29.84, 29.81, 29.75, 29.71, 29.59, 29.56, 29.52, 29.36, 29.32, 29.14, 27.08, 24.86, 22.86, 14.19; HRMS m/z calcd for C24H43O2F3Na

	H
	NMR (400 MHz, CDCl 3 ) d (ppm) 5.44-5.30 (2 H, m), 4.45 (2 H, q, J H-F = 8.5 Hz), 2.40 (2 H,
	t, J= 7.5 Hz), 2.06-1.91 (4 H, m), 1.72-1.59 (2 H, m), 1.39-1.15 (28 H, m), 0.87 (3 H, t, J= 6.8

  .56, 32.45, 32.01, 31.79, 29.62, 29.55, 29.38, 29.29, 27.43, 27.39, 27.07, 22.81, 22.71, 14.18, 14.12; HRMS m/z calcd for C 18 H 33 O 2 F 3 Na [M+Na] + : 361.2325, found: 361.2329.

	2,2,2-trifluoroethyl 9,12-dicyclopropylstearate 2f

  71.31, 71.09, 70.71, 63.85, 62.24, 34.66, 32.12, 30.21, 29.94, 29.80, 29.72, 27.74, 26.16, 25.47, 23.13, 14.31; HRMS m/z calcd for C 30 H 52 O 12 Na [M+Na] + : 627.3351, found: 627.3350. CDCl 3 /CD 3 OD: 1:1) d (ppm) 174.65, 130.27, 130.08, 128.14, 127.98, 93.60, 73.14, 71.71, 70.36, 70.14, 63.27, 34.19, 31.60, 29.71, 29.42, 29.31, 29.24, 29.22, 27.28, 27.27, 25.70, 24.92, 22.65, 14.06; HRMS m/z calcd for C 48 H 82 O 13 Na [M+Na] + : 889.5648, found: 889.5646. NMR (100 MHz, CD 3 OD): δ C 177.95, 95.08, 74.58, 73.17, 71.99, 71.94, 71.53, 64.13, 64.06, 54.80, 32.99, 32.96, 32.14, 30.75, 30.70, 30.08, 26.63, 26.59, 23.69, 23.67, 14.35, 14.33, 12.21, 12.18; HRMS m/z calcd for C 28 H 50 O 13 Na [M+Na] + : 617.3144, MeOH); 1 H NMR (400 MHz, CD 3 OD) d (ppm) 5.06 (2H, d, J = 3.7 Hz), 4.37 (2H, dd, J = 11.8, 2.1 Hz), 4.21 (2H, dd, J = 11.8, 5.3 Hz), 4.02 (2H, ddd, J = 10.1, 5.3, 2.2 Hz), 3.80 (2H, dd, J = 9.7, 8.8 Hz), 3.46 (2H, dd, J = 9.7, 3.8 Hz), 3.39-3.31 (1H, m), 2.36 (2H, tt, J = 9.0, 5.2 Hz), 1.65-1.55 (4H, m), 1.53-1.41 (5H, m), 1.29 (45H, m), 0.94-0.86 (12H, m); 13 C NMR (100 MHz, CD 3 OD) d (ppm) 178.08, 95.01, 74.59, 73.21, 72.02, 71.42, 64.23, 47.14, 33.65, 33.06, 32.89, 32.86, 30.70, 30.69, 30.60, 30.58, 30.45, 30.38, 30.37, 28.54, 28.52, 28.49, 28.47, 23.77, 23.70, 14.50, 14.48; HRMS m/z calcd for C 44 H 82 O 13 Na [M+Na] + : 841.5648, found: 841.5658. The general procedure has been applied to the synthesis of diester 4f. The titled compound was isolated as a colorless oil in 82% yield. [α] 20 D +4.6 (c 1.0; MeOH); 1 H NMR (400 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 4.91 (2H, d, J = 3.5 Hz), 3.77 (2H, dt, J = 7.7, 2.3 Hz), 3.61-3.53 (2H, m), 3.47 (3H, d, J = 3.3 Hz), 3.37-3.29 (2H, m), 3.24-3.12 (8H, m), 2.16 (4H, dd, J = 8.7, 6.3 Hz), 1.42 (4H, s), 1.28-1.02 (38H, m), 0.76-0.63 (8H, m), 0.59 (4H, m), 0.49 (4H, s), 0.41 (4H, m), (-0.39)-(-0.59) (4 H, m); 13 C NMR (100 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 174.94, 93.94, 77.89, 73.70, 72.00, 70.68, 70.36, 63.55, 34.45, 32.20, 30.45, 30.18, 29.77, 29.64, 29.46, 29.19, 29.03, 28.35, 28.20, 25.18, 22.99, 16.34, 16.21, 16.17, 15.94, 15.90, 14.23, 11.24, 11.07, 11.06; HRMS m/z calcd for C 52 H 90 O 13 Na [M+Na] + : 945.6274, found: 945.6283.

	6,6'-di-(dicyclopropyl)-stearoyl trehalose 4f
	6,6'-diethylhexanoyl trehalose 4d
	The general procedure has been applied to the synthesis of monoester 4d (n-hexane/t-BuOH
	6,6'-dilinoleoyl trehalose 4a was used instead of t-BuOH alone). The titled compound was isolated as a white solid in 38%
	The general procedure has been applied to the synthesis of diester compound 4a. The titled yield. mp: 181-183 °C; [α] 20 D +4.7 (c 1.0; MeOH); 1 H NMR (400 MHz, CD 3 OD) d (ppm) 5.05
	m a n u s c r i p t compound was isolated as a white solid in 85% yield. mp: 121-123 °C; [α] 20 D +5.9 (c 1.0, MeOH); 1 H NMR (400 MHz, CDCl 3 ) d (ppm) 5.31-5.13 (8H, m), 4.97 (2H, d, J = 3.7 Hz), 4.24-4.10 (4H, m), 4.00 (7H, s), 3.85 (2H, ddd, J = 10.1, 4.7, 2.5 Hz), 3.75 (2H, t, J = 9.4 Hz), 3.42 (2H, dd, J = 9.8, 3.7 Hz), 3.31-3.19 (2H, m), 2.68-2.60 (3H, m), 2.21 (4H, t, J = 7.6 Hz), 1.92 (7H, d, J = 6.9 Hz), 1.48 (4H, m), 1.29-1.08 (29H, m), 0.76 (6H, t, J = 6.9 Hz); 13 C NMR (100 MHz, 6,6'-dioleoyl trehalose 4b m a n u s c r i p t (2H, dd, J = 3.7, 1.5 Hz), 4.41 (2H, dd, J = 11.8, 2.1 Hz), 4.21 (2H, dd, J = 11.9, 5.2 Hz), 4.01 (2H, ddd, J = 10.1, 5.2, 2.1 Hz), 3.79 (2H, dd, J = 9.8, 8.8 Hz), 3.46 (2H, ddd, J = 9.8, 3.8, 1.4 Hz), 3.40-3.32 (2H, m), 2.30 (2H, tt, J = 8.9, 5.3 Hz), 1.70-1.42 (8H, m), 1.42-1.22 (14H, m), 0.95-0.84 (12H, m); 13 C found: 617.3155. 6,6'-dihexyldecanoyl trehalose 4e The general procedure has been applied to the synthesis of monoester 4e (n-hexane/t-BuOH m a n u s c r i p t
	α] 20 CDCl 3 /CD 3 OD : 1:1) d (ppm) 5.40-5.23 (4H, m), 5.09 (2H, dd, J = 12.0, 3.7 Hz), 4.33 (1H, dd, D +7.3 (c 1.0; MeOH); 1 H NMR (400 MHz, J =12.0, 2.2 Hz), 4.23 (1H, dd, J = 12.0, 4.9 Hz), 3.99 (1H, ddd, J =10.2, 4.8, 2.2 Hz), 3.87-3.74 (4H, m), 3.72-3.61 (2H, m), 3.50 (2H, td, J = 10.2, 3.7 Hz), 3.42-3.27 (4H, m), 2.74 (2H, t, J = 6.3 Hz), 2.33 (2H, t, J = 7.5 Hz), 2.03 (4H, dt, J = 8.5, 6.4 Hz), 1.59 (2H, q, J = 7.3 Hz), R e v i s e d The general procedure has been applied to the synthesis of diester 4b. The titled compound was isolated as a yellowish solid in 91% yield. mp: 153-154 °C; [α] 20 D +6.5 (c 0.8; MeOH); 1 H NMR (400 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 5.40-5.24 (4H, m), 5.07 (2H, d, J = 3.8 Hz), 4.32 (2H, dd, J = 12.0, 2.3 Hz), 4.23 (2H, dd, J = 12.0, 5.1 Hz), 3.96 (2H, ddd, J = 10.2, 5.1, 2.3 Hz), 3.75 (2H, dd, J = 9.7, 8.9 Hz), 3.49 (2H, dd, J = 9.7, 3.8 Hz), 3.38-3.33 (4H, m), 2.32 (4H, t, J = 7.5 Hz), 2.08-1.89 (7H, m), 1.60 (4H, m), 1.39-1.16 (39H, m), 0.86 (6H, t, J = 6.9 Hz); 13 C NMR (100 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 175.15, 130.48, 130.26, 94.33, 73.96, R e v i s e d was used instead of t-BuOH alone). The titled compound was isolated as a white solid in 33% yield. mp: 164-166 °C; [α] 20 D +5.4 (c 1.0; R e v i s e d
	1.39-1.21 (14H, m), 0.87 (3H, t, J = 6.8 Hz); 13 C NMR (100 MHz, CDCl 3 /CD 3 OD : 1:1) d 72.36, 71.08, 70.71, 63.88, 34.68, 33.13, 32.48, 30.31, 30.27, 30.25, 30.21, 30.18, 30.07, 30.05,
	(ppm) 175.17, 130.61, 130.48, 128.62, 128.51, 94.54, 94.41, 73.94, 73.72, 73.11, 72.47, 72.40,

29.92, 29.89, 29.84, 29.83, 29.76, 29.70, 29.67, 27.72, 27.69, 25.46, 23.21, 23.19, 14.34, 14.32; HRMS m/z calcd for C 48 H 86 O 13 Na [M+Na] + : 893.5961, found: 893.5969. 6-linoleoyl-6'-oleoyl trehalose 5

  1, 2.2 Hz), 3.43 (2H, dd, J = 9.7, 8.9 Hz), 3.16 (2H, dd, J = 9.7, 3.8 Hz), 3.05-2.94 (4H, m), 2.47-2.34 (2H, m), 1.99 (4H, t, J = 7.6 Hz), 1.75-1.64 (8H, m), 1.26 (4H, m), 1.06-0.89 (38H, m), 0.58-0.48 (6H, m); 13 C NMR (100 MHz, CDCl 3 /CD 3 OD : 1:1) d (ppm) 175.15, 130.61, 130.48, 130.47, 130.27, 128.62, 128.50, 110.72, 94.37, 73.96, 72.39, 71.11, 70.74, 63.90, 34.69, 32.51, 32.12, 30.33, 30.29, 30.20, 30.09, 29.94, 29.91, 29.86, 29.78, 29.70, 27.74, 26.16, 25.47, 23.23, 23.13, 14.35, 14.32; HRMS m/z calcd for C 48 H 84 O 13 Na [M+Na] + : 891.5804, found: 891.5815.

Acknowledgements

We are grateful to IFCPAR/CEFIPRA organization for a fellowship attributed to S. P. (Grant N°5105-1). We also thank the CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche for financial supports.


Léo Plaçais

Arsène Mekinian

Marie Bornes

Armelle Poujol-Robert

Naiké Bigé

Olivier Fain

Arsene Mekinian
email: arsene.mekinian@aphp.fr

Hôpital Saint-Antoine

Adult's onset Still disease occurring during pregnancy: case-report and literature review

Keywords: AOSD, pregnancy, pregnancy-revealed AOSD Conflicts

Pregnancy-revealed AOSD is a specific subset with

Article

SIR,

Adult onset Still's disease is a rare affection classified among non-hereditary autoinflammatory diseases. The clinical presentation goes from fever with arthralgia and maculopapular eruption to life-threatening manifestations such as secondary lymphohistiocytosis. The mechanisms of AOSD remains unclear, but seems implicate NK cells dysfunction with pro-inflammatory cytokines secretion including IL-1ß, IL-6 and IL-18 [START_REF] Avaua | Cytokine balance and cytokine-driven natural killer cell dysfunction in systemic juvenile idiopathic arthritis[END_REF]. Several reports mentioned AOSD occurring during pregnancy, mostly in first and second trimester and there is still a debate on whether it could compromise or not the pregnancy outcome. We here report a case of AOSD revealed during pregnancy with a life-threatening presentation along with a review of 19 cases from literature.

A 38-years old woman was treated in our department for diffuse systemic sclerosis and associated Sjögren syndrome. Both diseases were quiescent at the time of the report and none required immunosuppressive therapy. In May 2016 she was pregnant at 12 weeks of gestation and presented with acute fever without any associated features. Laboratory data revealed mild liver cytolysis (AST and ALT x4N) but a large screening for infectious and auto-immune diseases was negative and hepato-biliar imaging was normal. Fever and cytolysis spontaneously regressed in 3 days. Two month later, she was hospitalized again for intense fever, sore throat and abdominal pain.

Laboratory analysis revealed liver cytolysis (ALT and AST x10N) with cholestasis, elevated bilirubin and increased C-reactive protein levels (53mg/L). Ferritin plasma levels were slightly high (657 ng/mL). Extended screening for infection remained negative and symptoms spontaneously regressed and she was discharged without any treatment. One month later she was admitted again for fever and arthromyalgia at 28 weeks of gestation. Laboratory data showed increased C-reactive protein levels (163 mg/L) associated with liver cytolysis (x2N), and ferritin levels at 371 mg/l. Cervico-thoraco-abdominal imaging revealed mild non-compressive pericarditis and persistent homogenous hepatomegaly (22cm of great axis). Because of persistent fever and polyarthralgias, after exclusion of active infection, steroids were started (prednisone 1 mg/kg) associated with colchicine, which allowed clinical remission and C-reactive protein significant decrease. When decreasing steroids at 12.5 mg/day, she again experienced fever and arthralgias recurrence and intravenous immunoglobulins were started. Just before intravenous immunoglobulins, acute agranulocytosis occurred and was considered related to colchicine. The agranulocytosis was complicated with severe sepsis from urinary tractus infection, so foetal extraction was decided and realized at 34 weeks of gestation. Despite foetal extraction and sepsis control, the fever and polyarthralgias persisted, associated with severe cholestasis (x15N) and C-reactive protein levels at 320 mg/l. Ferritin levels were thus at 41 000 ng/mL with glycosylated ferritin less than 5%. The diagnosis of AOSD was stated according to Yamaguchi and Fautrel criteria [2 ; 3] and steroids were associated with anakinra (100 mg/day) allowing rapid clinical and biological remission.

AOSD can be revealed by pregnancy as first suggested in a case report from 1982 [START_REF] Green | Adult Still's disease associated with pregnancy[END_REF] eleven years after the first disease's description. We gathered data about 19 additional cases from the literature of AOSD revealed during the pregnancy [Table 1]. All cases concerned women from 19 to 38 years old (median age 28.6). Upon 18 pregnancies, median term at first flare was 16.77 weeks of gestation. Most women were in their first and second trimester (from 8 to 26 weeks of gestation).

Clinical features mostly included arthritis and arthromyalgia, hectic fever and pharyngitis. Ferritin levels ranged from 1311 to 41 424 ng/mL. Most patients required steroids (n=16/20), and two were treated by intravenous immunoglobulins. All patients had systemic involvement and none presented as a chronic articular form. There were no obvious differences between the monocyclic and polycyclic forms. Obstetrical complications were frequent (n=11/20) mostly showing up as prematurity (n=10/20) though ours was due to foetal extraction and three of them could be linked to pre-term premature rupture of membranes potentially induced by the steroid regimens. There were three cases with intrauterine growth restriction, two with oligohydroamnios and one with neonatal death. In pregnancy-revealed AOSD, flares seemed to appear in first and second trimester earlier than in known AOSD [START_REF] Gerfaud-Valentin | Adult-onset Still's disease and pregnancy: about ten cases and review of the literature[END_REF]. Obstetrical complications seemed to be frequent as 50% of the pregnancies ended up with prematurity and 15% complicated by intra-uterine growth restriction even though some could be related to steroid therapy adverse events. The pathophysiological link isn't clear but recent studies showed high levels of IL18 during pregnancy [START_REF] Wang | Metabolic profiling of pregnancy: cross-sectional and longitudinal evidence[END_REF] that could act as a trigger to a previously latent disease and be responsible for pregnancy-induced AOSD. The treatment of AOSD during pregnancy could be challenging. Steroids can be used, but may be associated with adverse events, like gestational diabetes, arterial hypertension. Intravenous immunoglobulins can be used if necessary in presence of life-threatening AOSD [START_REF] Liozon | Intravenous immunoglobulins for adult Still's disease and pregnancy[END_REF]. The use of other agents, like anakinra and tocilizumab cannot be recommended during pregnancy [START_REF] Levy | Critical review of the current recommendations for the treatment of systemic inflammatory rheumatic diseases during pregnancy and lactation[END_REF]. In conclusion, pregnancy-revealed AOSD appears to be a specifical subset of the disease with a systemic course, flares on first and second trimester, obstetrical complications such as prematurity and IUGR sometimes leading to life-threatening situations requiring parenteral corticotherapy and intravenous immunoglobulins.

  Wg : weeks of gestation ; A : arthritis, E : erythema, F : fever, L : lymphadenopathy, S : seritis, AM : arthromyalgia, FE : foetal extraction, Pha : pharyngitis, OHA : oligohydroamnios, SMG : splemomegalia, HSMG : hepatosplenomegalia, IUGR : intrauterine growth restriction, LCAP : leucocytapheresis, PPRM : preterm premature rupture of membranes, Iv IG : intravenous immunoglobulins, PP : post partum.

Table 1 . AOSD revealed during pregnancy: cases from the literature review.

 1

	Year /	Age	Time of	AOSD	Ferritin	Treatment	AOSD	Adverse
	Author		pregnancy	features	levels /		outcome	Obstetrical
			(wg)		GF levels		during the	outcome
							pregnancies
	1980,	34	Unknown A, F, LN,	NA/NA Aminopyrin	Polycyclic	None
	Kaplinsky [1]			E		e, gold salt	(PP)
	1982, Green	23	25 wg	A,F,	NA/NA	NA	Polycyclic	Neonates
	[2]			HSMG,			(28wg, PP)	death,
				LN, E				prematurity
								at 28wg
	1985, Yebra	19	12 wg	A,F,	NA/NA	Prednisone Monocyclic	0
	Bango [3]			HSMG,
				E, Pha
	1990, Katz [4]	32	PP 2 m	E, A	NA/NA	NSAID	Monocyclic	0
	1993, Le Loët	27	20 wg	A, F, L,	NA/NA	Prednisone	Polycyclic	OHA
	[5]			E, Pha,		1 mg/kg/d
	1993, Le Loët	24	20 wg	A, F, L,	NA/NA	Prednisone	Polycyclic	0
				E, Pha,		20 mg/d
				AM
	1994,	25	8 wg	A, F, L,	NA/NA	High dose	Polycyclic	FE
	Falkenbach			AM,		prednisone,	(suites de
	[6]			HSMG,		EDX	couches)
				S, Pha,


Irène Nicolas

Olivier Jeannin

Delphine Pichon

Marc Fourmigué

Dibromohydantoins as halogen bond (XB) donors: a route toward the introduction of chirality in halogen bonded systems †

Dibromohydantoins as halogen bond (XB) donors: a route toward the introduction of chirality in halogen bonded systems

Introduction

The formation of chiral supramolecular assemblies is an attractive challenge in the context of crystal engineering, 1,2 It most often relies on the use of different intermolecular interactions such as metal coordination, hydrogen bonding 3 or π-π interactions. 4 By comparison, the use of halogen bonding 5 (XB) as a crystal engineering tool in the formation of chiral, eventually helical structures has been only rarely mentioned, albeit XB has been also recently established as a powerful intermolecular interaction for the elaboration of complex supramolecular architectures. [START_REF] Desiraju | For the IUPAC definition of halogen bond[END_REF][START_REF]Halogen Bonding: Fundamentals and Applications[END_REF]8,9 One of the earliest examples is the resolution of racemic 1,2dibromohexafluoropropane through co-crystallisation with (-)-sparteine hydrobromide into supramolecular helices stabilised by C-Br•••Br -XB interactions. 10 Halogen bonds can be also associated with hydrogen bonding, for example in chiral amines, 3b ammoniums salts 11 or sulfinamides. 12 Chirality has been introduced in conducting halogen bonded tetrathiafulvalenium salts, through chiral counter ions 13 or chiral iodinated TTF themselves. 14 One can also mention chiral nematic phases from halogen-bonded, bent-core achiral mesogen, 15 host-guest compounds involving octafluorodiiodobutane, 16 asymmetrical porphyrin derivatives, 17 or chiral anion recognition. 18 In order to favour the formation of intrinsically chiral structures based on halogen bond interactions, we are currently investigating neutral iodinated spacers with a built-in chirality and recently described ditopic halogen bond donors derived from chiral diols (Scheme 1a). 19 Their crystallisation with halide anions as halogen bond acceptors afforded helical halogen bonded anionic chains.

Looking for more compact but still chiral ditopic halogen bond donors, we turned our attention to N-iodoimides derivatives, known to act powerful halogen bond donors. 20 For example, N-iodosuccinimide, 21,22 or N-iodosaccharin, 23 originally used as electrophilic iodinating agents can be efficiently engaged in XB systems, for example with pyridines of various Lewis base character. 24 In the same vein, N,N'-diodo-dimethylhydantoin (DIH) has found many uses in organic synthesis as halogenating agents 25 or as catalysts. 26 It can also act as a powerful halogen bond donor with both an imidic and an amidic N-I halogen bond donor groups (Scheme 1b). We have recently demonstrated, in the halogen bonded adducts of DIH with pyridines, an anti-cooperativity property: while the most basic pyridines were able to cocrystallize with DIH into bis-adducts where both iodine atoms are indeed involved in XB interactions, the less basic ones led to monoadducts involving only the imidic N-I moiety. The hydantoin core offers another attractive feature associated with the substitution pattern at 5-position. Two methyl groups are found in the prototypical dimethylhydantoin and its diiodo derivative diiododimethylhydantoin (DIH) but the presence of two different substituents at the 5-position affords indeed a chiral hydantoin (Scheme 1b). Many of them have been in fact reported in the literature for therapeutic applications, 28 most of them derived from natural amino acids. On the other hand, the corresponding N,N'-dihalo derivatives of such chiral hydantoins were only scarcely mentioned. Chiral dichloro hydantoins are mentioned as a chlorenium source in asymmetric chlorolactonization reactions, 29 while dibromo derivatives were only mentioned as racemic mixtures as CCl4-soluble halogenating agents. 30 Chiral N,N'-diiodo derivatives are still unknown. In the following, we have explored the preparation of four different chiral N,N'-dibromohydantoin derivatives (1-R in Scheme 2) and their association, together with that of achiral 5,5-dimethyl-N,N'-dibromo derivative DBH, with pyridines of various Lewis base character. We were able to isolate single crystalline phases from the association of DBH with electron-poor pyridines such as 4cyanopyridine, 4-trifluoromethylpyridine and methylisonicotinate. Among the chiral dibromohydantoins prepared here, the 1-iBu dibromohydantoin derived from leucine was also successfully co-crystallized, in a 1:1 stoichiometry, with methylisonicotinate.

Results and discussion

Syntheses

The main synthetic route toward chiral hydantoins is based on the formation of an urea 2-R from the starting amino acid methyl ester (Scheme 3). Further cyclization to the desired hydantoins 3-R can been then performed, either under acidic 31 or basic conditions. 32 This second step is often difficult because affected by racemisation problems. As shown in Scheme 3, we have use here the methylester derivatives of four different amino acids, phenylglycine (R = Ph), phenylalanine (R = Bz), valine (R = iPr) and leucine (R = iBu). The ureido intermediates 2-R were formed in good yields (67 to 74 %) from the reaction of the amino acid methylester hydrochloride with potassium cyanate in water at room temperature. 33 Being aware of the racemisation problems reported earlier, an optimization of the cyclization conditions was conducted on the phenylalanine ureido derivative (R = Bz). Conducting the cyclization step in basic medium with sterically hindered amines such as DMAP or NEt3 as reported, led however to the racemised hydantoins, as shown by HPLC. A similar optimization of the cyclisation step, but under acidic conditions, has been also reported by Dakin et al,31b by varying the experimental conditions (nature, concentration of acid and reaction time) and monitoring the optical activity of the final hydantoin product. Based on this study, we used 2N HCl and a minimum reflux time, allowing for a complete conversion of the four different ureas 2-R to the corresponding hydantoins 3-R (in 59-89% yields) while maintaining the initial optical purity.

The next dibromation step was performed as described elsewhere 34 for achiral hydantoins, by neutralization of 3-R with two equiv. KOH and reaction with dibromine at 10°C. The dibromohydantoins 1-R were obtained in excellent yields (90-94%) but only with R = Ph, iPr and iBu, while 5-benzylhydantoin 3-Bz was degraded under these conditions.

Halogen bonding in the pyridine adducts

The co-crystallisation of achiral DBH with the electron-rich pyridines such as pyridine itself, 4-picoline, or 4-dimethylaminopyridine did not afford the expected adducts. Bromine-free dimethylhydantoin was recovered. With picoline, we could only isolate crystals of 4methylpyridinum bromide. On the other hand, the pyridines substituted in para position with electron-withdrawing substituents (-CN, -CF3, -CO2Me) afforded systematically a 1:1 adduct (Fig. 1), despite the presence of two equivalents pyridine in the solution before crystallisation by vapour diffusion. DBH•[Py-CO2Me] crystallises in the triclinic system, space group 1 , with one halogen-bonded monoadduct in general position in the unit cell (Fig. 1a). The imidic bromine atom is halogen bonded to the nitrogen atom of methylisonicotinate while the other bromine atom in amidic position is halogen bonded to one oxygen atom of a neighbouring molecule.

Note the disorder of the ester group on two positions with a 75:25 distribution. Relevant bond distances for these two intermolecular interactions are collected in Table 1, and

Lewis base

Intramol. dist. The evolution of the halogen bond distances with the nature of the Lewis base XB acceptor is particularly interesting to discuss here. We first note that the stronger XB interaction involves the imidic bromine atom, as already observed in the analogous diiodohydantoin analog. 27 The interaction is particularly strong, with RR values in the range 0.68-0.72. Also, the interaction is stronger, i.e. the reduction ratio RR is smaller, with the more electron rich pyridines. The observed ranking, Py-CO2Me > Py-CF3 > Py-CN > Pyrazine is fully consistent with our own earlier observations with diiodohydantoin adducts, 27 as well as with other pKB scales established for examples with diiodine (I2) as XB donor. 35 The XB interaction also affects the dibromohydantoin itself as the intramolecular N-Brimidic distance increases as the bromine is can be obtained from the comparison of the reduction ratios. As shown in Table 1, the RR(Br,N) and RR(I,N) are essentially the same, 0.678 vs. 0.679, in contradiction to the general rule that states that halogen bonding is systematically stronger with iodine than with bromine. A similar behaviour has been mentioned by Rissanen et al. 36 in the haloniumpyridine [Py-X-Py] + cations, 37,38 where the reduction ratio for the N As mentioned in the Introduction, the interest of such diodo-or dibromohydantoins also lies in their ability to potentially provide chiral, enantiopure ditopic halogen bond donors.

N-Brimidic (Å) Brimidic•••NPy dist. (Å) RR(BrN) Intramol. dist. N-Bramidic (Å) Bramidic•••Y dist. (Å) RR(
For that purpose, the chiral hydantoins 3-R derived from (S)-phenylglycine (R = Ph), phenylalanine (R = Bz), valine (R = iPr) and leucine (R = iBu) were prepared and the corresponding dibromo derivatives 1-R were isolated with, as substitution pattern on the 5 position, one hydrogen and one R group, R = Ph, iPr and iBu. Note that 1-Bz could not be isolated under these conditions. Our first attempts to co-crystallise the three novel chiral

Experimental

General. 1 H and 13 C spectra were collected on a BRUKER AV 300 ( 1 H : 300 MHz, 13 C : 75,5 MHz). Chemical shifts δ are expressed in part per million (ppm), vs. the deuterated solvent used as reference, such as d [START_REF] Desiraju | For the IUPAC definition of halogen bond[END_REF] -DMSO ( 1 H NMR δ 2.50 ppm, 13 C NMR δ 128.06 ppm).

The following abbreviations were used to describe signal multiplicity: bs broad singlet, s General procedure for the preparation of the chiral ureas (2-R). In a 100 mL flask, the aminoester hydrochloride (2 g) is dissolved in water (170 equiv.) and KOCN (1.02 equiv.) is added. The mixture is stirred for one hour at room temperature. [α]D (c = 0.98.10 -3 g mL -1 , EtOH) = +103.06° (Litt [39] : +112°, c = 0.20 g.mL -1 , EtOH).

2-Ph

3-Bz.

With 2-Bz (1.013 g, 4.6 mmol) and HCl 2N (19 mL, 8.3 equiv.). The solution is extracted with AcOEt. The combined organic phases are dried over Na2SO4, filtered and concentrated to dryness. Yield: 764 mg (87 %). M.p. 175-176 °C (Litt. [30a] 187-189 °C). [α]D (c = 0.97.10 -3 g mL -1 , EtOH) = -98.97° (Litt [40] -96°; c = 0.20 g.mL -1 , EtOH).

3-iPr.

With 2-iPr (1.047 g, 6.0 mmol) and HCl 2N (30 mL, 8.3 equiv.). The solution is extracted with AcOEt. The combined organic phases are dried over Na2SO4, filtered and concentrated to dryness. Yield: 688 mg (81 %). M. p. 131-132 °C (Litt. [31a] 144-146 °C). [α]D (c = 1.08.10 -3 g mL -1 , EtOH) = -94.44° (Litt. [40] -94°, c = 0.20 g.mL -1 , EtOH)

3-iBu.

With 2-iBu (1.002 g, 5.3 mmol) and HCl 2N (32 mL, 8.3 equiv.). The filtered residue is washed with cold water and dried under vacuum over KOH. Yield: 674 mg (81%). M.p.

213-214 °C (Litt. [31a] 213-214 °C). 1 H NMR (DMSO-d6, 300 MHz) δ 0.88 (2d, 3 J1 = 6.6 Hz, [α]D (c = 1.07.10 -3 g.mL -1 , EtOH) = -78.5°.

General procedure for the preparation of the N,N'-dibromohydantoins (1-R). In a 100 mL flask, hydantoin 3-R (200 mg) is suspended in water (111 equiv.) and the suspension cooled to 10°C. NaOH (1N in water, 2 equiv.) is added and the mixture stirred until complete dissolution. The flask is covered to work in the dark and Br2 (2 equiv.) is added dropwise.

After stirring for 20 min at 10°C, the resulting precipitate is filtered, washed with cold water and dried in vacuum over KOH. Crystallography. X-ray crystal structure collections were performed on an APEXII Bruker-AXS diffractometer equipped with a CCD camera and a graphite-monochromated Mo-Kα radiation source (λ = 0.71073 Å). Details of the structural analyses are summarized in Table 2. Absorption corrections were performed with SADABS. Structures were solved by direct methods using the SIR97 program, 41 and then refined with full-matrix least-squares methods

1-

27

 27

  Scheme 1(a) Chiral ditopic XB donors reported from chiral diols.19 (b) Diodo-and dibromohydantoins.

Scheme 2

 2 Scheme 2 Dibromohydantoins investigated with pyridines as XB acceptors.

Scheme 3

 3 Scheme 3 Synthetic path to the chiral dibromohydantoins.

Fig. 1 .

 1 Fig. 1. Detail of the halogen bond interactions in the 1:1 adducts between DBH and respectively (a) methyisonicotinate, (b) 4-trifluoromethylpyridine and (c) 4-cyanopyridine.

  compared with the sum of the van der Waals radii (rX, rY) of interacting atoms (X, Y) to define a socalled reduction ratio (RR), i.e. d(X•••Y)/(rX + rY). DBH•[Py-CF3] crystallises in the monoclinic system, space group P21/c with one XB monoadduct in general position in the unit cell (Figure 1b). Intermolecular interactions parallel those observed above with a strong Br•••NPy interaction involving the imidic bromine atom and a weaker Br•••O interaction with the amidic bromine atom. The situation is slightly different in DBH•[Py-CN]. It crystallises in the monoclinic system, space group C2/m with the XB adduct located on a mirror plane containing the hydantoin core while the cyanopyridine is perpendicular to this plane. This adduct crystallises with one 1,2-dichloroethane molecule, located on a 2/m position, hence the exact formulation DBH•[Py-CN]•(ClH2C-CH2Cl)0.5. Furthermore, besides the strong Br•••NPy halogen bond involving the imidic bromine atom, the other amidic bromine atom is not halogen bonded to an oxygen atom as observed above with Py-CO2Me and Py-CF3, but is almost at van der Waals contact with a neighbouring bromine atom. The corresponding reduction ration for this Br•••Br interaction amounts indeed to 0.985. At last, the pyrazine adduct involves two DBH molecules for one pyrazine, located in an inversion centre. Halogen bonding Br•••N interactions involve the imidic bromine atom while the amidic bromine is bonded to the oxygen atom of a neighbouring DBH molecule (Fig. 2).

Fig. 2

 2 Fig. 2 Detail of the halogen bond interactions (blue and red dotted lines) in the adduct (DBH)2•Pyrazine.

  s c r i p t involved in the strongest XB. Another interesting feature is the fact that the second, amidic bromine atom is not halogen bonded to a second pyridine (despite an excess of pyridine in the crystallisation process) but is halogen bonded to the carbonyl oxygen atom of a neighboring DBH molecule. This behaviour was already encountered with diodohydantoin (DIH) faced with these electron poor pyridines.27 It is associated to a strong polarization of the XB adduct following the setting of the strongest XB through the imidic bromine atom (Scheme 4). The partial negative charge which delocalizes on the DBH moiety deactivates the second amidic bromine atom while it increases simultaneously the negative charge of the carbonyl oxygen atoms. Last but not least, a comparison of the strength of the halogen bond, within the bromo and iodo adducts of methylisonicotinate, that is DBH•[Py-CO2Me] and DIH•[Py-CO2Me]

  Scheme 4 Polarization on the XB adduct DBH•[Py-R].

  with different pyridines afforded a crystalline phase from the association of the 1-iBu with methylisonicotinate, formulated as [1-iBu]•[Py-CO2Me]. It crystallises in the orthorhombic system, chiral space group P212121, with one adduct in general position in the unit cell (Fig. 3). The halogen bonded motifs parallels those observed above with the achiral DBH, with the imidic bromine atom engaged in XB with the pyridinic nitrogen while the other amidic bromine atom links to the carbonyl oxygen atom of a neighbouring molecule by translation along the b axis. The structural characteristics of the strongest Br•••NPy XB interaction (Table 1) are very close to those observed within the adduct of achiral DBH with the same Lewis base, methylisonicotinate, but the secondary Br•••O interaction is notably weakened (2.898 Å instead of 2.802 Å). It is associated with a lowered directionality, with the Br atom lying 1.31 Å above the basal plane defined by the neighbouring heterocycle along b, while within the adduct of achiral DBH, this bromine atom is only at 0.36 Å above the basal plane of the neighbouring molecule.

Fig. 3

 3 Fig. 3 Detail of the halogen bond interactions in the [1-iBu]•[Py-CO2Me] adduct.

  singlet, d doublet, t triplet, q quadruplet, m multiplet, dd doublet of doublet, ddd doublet of doublet of doublet. Analytical thin layer chromatography was performed with MACHEREY-NAGEL 60F254 plates (Silica on aluminum foil 60F254, 60/20 mm) and the plates were revealed by Ninhydrin solution and read with UV fluorescence at 254 nm. Chiral HPLC was performed on a Varian Prep Star 218 equipped with Photodiodes Varian Pro Star 330. Ananalytical Chiracel® OD-H (4,6x250 mm, 5 µm) column was used with as eluent an hexane/isopropanol mixture, at 1 mL.min -1 flow with solutions of 5 mg sample for 500 µL isopropanol. Absolute optical rotations [α]D were determined on a Perkin Elmer 341 polarimeter using the Na line at 589 nm according to = × where is αexp is the observed rotation angle, l the length of optical path (10 cm) and c the concentration (in g.mL -1 ).

  1 H NMR (DMSO-d6, 300 MHz) δ 0.80 (d, 3 J = 6 Hz, 3H, CH3), 0.94 (d, 3 J = 6 Hz, 3H, CH3), 1.98 (m, 1H, CH(CH3)2), 3.90 (dd, 3 J1 = 3 Hz, 3 J2 = 6 Hz, 1H, CHα), 7.89 (ls, 1H, CONH), 10.55 (ls, 1H, CONHCO). 13 C NMR (DMSO-d6, 75.5 MHz) δ 16.30 (CH(CH3)2), 18.99 (CH(CH3)2), 29.99 (CH(CH3)2), 63.19 (CHα), 158.26 (CONH), 175.87 (NHCONH). HPLC (Eluent Hexane/Isopropanol 90:10) τR = 10.541 min (ee 100 %).

3

  J2 = 4.8 Hz, 6H, 2CH3), 1.45 (m, 2H, CH2), 4.00 (ddd, 3 J1 = 9.3 Hz, 3 J2 = 4.5 Hz, 3 J3= 1.4 Hz,

  based on F 2 (SHELXL-97)42 with the aid of the WINGX program.43 All non-hydrogen atoms were refined with anisotropic atomic displacement parameters. H atoms were finally included in their calculated positions. Crystallographic data were deposited as CCDC 1509929: DBH•[Py-CN]•(C2H4Cl2)0.5; CCDC 1509930: (DBH)2•Pyrazine; CCDC 1509931: DBH•[Py-CO2Me]; CCDC 1509932: [1-iBu]•[Py-CO2Me] and CCDC 1509933: DBH•[Py-CF3].

  With 2-Ph (504.6 mg, 2.4 mmol) and HCl 2N (10 mL, 8.3 eq). The filtered residue is washed with cold water and dried under vacuum over KOH. Yield: 250 mg (59 %). M. p.

	2-Bz. L-Phenylalanine methyl ester hydrochloride (2 g, 9.97 mmol), KOCN (753 mg, 1.02 eq), H2O (30 mL, 170 equiv.). The solution is extracted with AcOEt. The organic phases are dried over Na2SO4, filtered and concentrated under reduced pressure. Yield: 1.36 g (67 %). 3-Ph. 177-178 °C (Litt [31a] 177-178 °C). 1 H NMR (DMSO-d6, 300 MHz) δ 5.16 (s, 1H, CH), 7.37
	M.p. 102-103 °C. 1 H NMR (DMSO-d6, 300 MHz) δ 2.92 (qd, 3 J1 = 13.7 Hz, 3 J2 = 6.8 Hz, 2H, (m, 5H, HAr), 8.39 (ls, 1H, CONH), 10.78 (ls, 1H, CONHCO). 13 C NMR (DMSO-d6, 75.5
	Ph-CH2), 3.60 (s, 3H, COOCH3), 4.38 (td, 3 J1 = 8.1 Hz, 3 J2 = 5.6 Hz, 1H, CHα), 5.63 (bs, 2H, MHz) δ (ppm) : 61.64 (CHα), 127.15 (CAr), 128.69 (CAr para), 129.10 (CAr), 136.50 (CAr),
	NH2), 6.28 (d, 3 J = 8.2 Hz, 1H, NH), 7.25 (m, 5H, HAr). 13 C NMR (DMSO-d6, 75.5 MHz) δ 157.94 (CONH), 174.64 (NHCONH). HPLC (Eluent Hexane/Isopropanol 80:20) τR = 15 min
	m a n u s c r i p t 38.06 (Ph-CH2), 52.10 (COOCH3), 54.33(CHα), 126.97 (CAr para), 128.70(CAr), 129.58 (CAr), 137.60 (CAr), 158.41 (COOCH3), 173.51 (CONH). HPLC (Eluent Hexane/Isopropanol 80:20) τR = 8.584 min. [α]D (c = 1.06.10 -3 g.mL -1 , EtOH) = +28.3°. 2-iPr. L-Valine methyl ester hydrochloride (2 g, 13 mmol), KOCN (973 mg, 1.02 equiv.), H2O (36 mL, 170 equiv.). The solution is extracted with AcOEt. The organic phases are dried over Na2SO4, filtered and concentrated under reduced pressure. Yield 1.63 g (74 %). M.p. 99-100 °C. 1 H NMR (DMSO-d6, 300 MHz) 0.85 (t, 3 J = 7.2 Hz, 6H, 2CH3), 1.96 (m, 1H, CH(CH3)2), 3.63 (s, 3H, COOCH3), 4.05 (dd, 3 J1 = 8.8 Hz, 3 J2 = 5.5 Hz, 1H, CHα), 5.61 (ls, 2H, NH2), 6,29 (d, 3 J = 8,8 Hz, 1H, NH). 13 C NMR (DMSO-d6, 75.5 MHz) δ 18.27 (CH(CH3)2), 19.43 (CH(CH3)2), 30.83 (CH(CH3)2), 51.92 (COOCH3), 58.0 (CHα), 158.86 (COOCH3), 173.64 (CONH). HPLC (Eluent Hexane/Isopropanol 90:10) τR = 10.8 min. [α]D m a n u s c r i p t (ee 99.7 %).
	. L-Phenylglycine methyl ester hydrochloride (2 g, 9.92 mmol), KOCN (845 mg, 1.02 eq), H2O (7 mL, 170 equiv.). The white precipitate is filtered and washed with cold water, and then dried over KOH in vacuum. Yield: 1.47 g (71 %). M.p. 136-137 °C. 1 H NMR (DMSO-d6, 300 MHz) δ 3.62 (s, 3H, COOCH3), 5.25 (d, 3 J = 6,0 Hz, 1H, CHα), 5.69 (ls, 2H, NH2), Hexane/Isopropanol 90:10) τR = 10.381 min. [α]D (c = 1,03.10 -3 g mL -1 , EtOH) = -17,47°. 6.86 (d, 3 J = 9Hz, 1H, NH), 7.36 (m, 5H, HAr). 13 C NMR (DMSO-d6, 75.5 MHz) δ 52.57 A c c e p t e d (c = 1.11.10 -3 g.mL -1 , EtOH) = -6.30°. 2-iBu. L-Leucine methyl ester hydrochloride (2 g, 12 mmol), KOCN (1.26 g, 1.02 equiv.), H2O (47 mL, 170 eq). The solution is extracted with AcOEt. The organic phases are dried over Na2SO4, filtered and concentrated under reduced pressure. Yield 1.53 g (68 %). M.p. 67-68 °C. 1 H NMR (DMSO-d6, 300 MHz) δ 0.87 (2d, 3 J1= 6.7 Hz, 3 J2= 6.6 Hz, 6H, 2CH3), 1.43 (t, 3 J = 7,5 Hz, 2H, CH2), 1.61 (m, 1H, CH(CH3)2), 3.61 (s, 3H, COOCH3), 4.14 (q, 3 J = 7,7 Hz, 1H, CHα), 5.55 (bs, 2H, NH2), 6.30 (d, 3 J = 8.2 Hz, 1H, NH). 13 C NMR (DMSO-d6, 75.5 MHz) δ 21.95 (CH(CH3)2), 23.19 (CH(CH3)2), 24.71 (CH(CH3)2), 41.24 (CH2) 51.21 (COOCH3), 52.07 (CHα), 158.63 (COOCH3), 174.71 (CONH). HPLC (Eluent A c c e p t e d
	(COOCH3), 57.12 (CHα), 127.66 (CAr), 128.52 (CAr para), 129.17 (CAr), 137.99 (CAr), 158.18

(COOCH3), 172.54

(CONH)

. HPLC (Eluent: Hexane/Isopropanol 80:20) τR = 8.376 min. [α]D (c = 9.9.10 -4 g mL -1 , EtOH) = +147.5°.

General procedure for the preparation of the chiral hydantoins (3-R).

The above prepared ureas 2-R are suspended in 2N HCl and stirred at reflux for one hour. 31b

  1H, CHα), 8.00 (ls, 1H, CONH), 10.57 (ls, 1H, CONHCO).13 C NMR(DMSO-d6, 75.5 MHz)

	δ 21.89 (CH(CH3)2), 23.58 (CH(CH3)2), 24.58 (CH(CH3)2), 41.24 (CH2), 56.54 (CHα),
	157.88 (CONH), 176.95 (NHCONH). HPLC (Eluent Hexane/Isopropanol 90:10) τR = 9.088
	min (ee: 99 %).

  Methylisonicotinate (17 µL, 9.56 10 -5 mol) was added. The solution was filtered to remove non-dissolved particles and poured in Durham tube. Crystals were made by vapor diffusion method. Hexane was used as precipitant. The sample was left to 2°C during 8 days in the dark. White crystals were obtained. M.p. 97-98°C. Elem. Anal. Calcd. for

	1-iBu. With 3-iBu (205 mg, 0.7 mmol), NaOH (2.6 mL, 2.6 mmol, 2 equiv.), H2O (2.6 mL, COOCH3), 1.39 (s, 6H, 2xCH3). Elem. Anal. Calcd. for C12H13Br2N3O4 (M = 423.061 g mol -
	0.14 mol, 111 equiv.) and Br2 (131 ìL, 2.6 mmol, 2 equiv.). The mixture is stirred at 10°C 1 ): C, 35.77; H, 2.67; N, 9.63 %. Found: C, 34.23; H, 3.04; N, 9.94 %.
	after Br2 addition for only 10 mn. Yield: 198 mg (90 %). M. p. 100-101 °C. 1 H NMR (ACN-
	d3, 300 MHz) 0.92 (d, 3 J = 6.6 Hz, 3H, CH3), 0.98 (d, 3 J = 6.6 Hz, 3H, CH3), 1.72 (m, 2H, (DBH)2(pyrazine). DBH (15 mg, 5.25 10 -5 mol) was dissolved in CH2Cl2 (2 mL). Pyrazine
	CH2), 4.24 (t, 3 J = 5.5 Hz, 1H, CH). 13 C NMR (ACN-d3, 75.5 MHz) δ (ppm) : 21.63 (21 µL, 15.75 10 -5 mol) was added. The solution was filtered to remove non-dissolved
	(CH(CH3)2), 21.99 (CH(CH3)2), 23.94 (CH(CH3)2), 38.43 (CH2), 66.90 (CH), 154.77 particles and poured in a Durham tube. Crystals were made by vapor diffusion method.
	(CONBr), 170.47 (NBrCONBr). [α]D (c = 9.6.10 -4 g.mL -1 , EtOH) = -8.3°. Hexane was used as precipitant. The sample was left at 2°C during 8 days in the dark. White
	Ph. With 3-Ph (195 mg, 0.92 mmol), NaOH 1N (2 mL, 2 mmol), H2O (2 mL, 0.11 mol, 111 equiv.) and Br2 (102 µL, 2 mmol, 2 equiv.). Yield: 304 mg (Yield 91%). M. p. 108-109 °C. 1 H NMR (ACN-d3, 300 MHz) δ 5.24 (s, 1H, CHα), 7.60 (m, 5H, HAr). 13 C NMR (ACN-d3, 75.5 MHz) δ 71,69 (CHα), 127,84 (CAr), 129,15 (CAr), 129,77 (CAr para), 132,48 (CAr), 154,45 (CONBr), 168,69 (NBrCONBr). Elem. Anal. Calcd for C9H6Br2N2O2 (M = 333.967 g mol -1 ) C, 32.37; H, 1.81; N, 8.39%. Found: C, 32.52; H, 2.41; N, 8.44%. [α]D (c = 1,12.10 -3 1-Bz. With 3-Bz (205 mg, 1 mmol), NaOH 1N (2.1 mL, 2.1 mmol, 2 eq), H2O (2.1 mL, 0.12 mol, 111 eq) and Br2 (108 L, 2.1 mmol, 2 eq). The reaction failed to provide tractable material. 1-iPr. With 3-iPr (200 mg, 1.4 mmol), NaOH (2.8 mL, 2.8 mmol, 2 eq), H2O (2.8 mL, 0.16 mol, 111 eq) and Br2 (144 µL, 2.8 mmol, 2 equiv.). Yield 450 mg (94 %). M. p. 114-115 °C. 1 H NMR (ACN-d3, 300 MHz) δ 0.96 (d, 3 J = 6.9 Hz, 3H, CH3), 1.09 (d, 3 J = 7.0 Hz, 3H, CH3), 2.26 (qd, 3 J1 = 7,0 Hz, 3 J2 = 3.2 Hz, 1H, CH(CH3)2), 4.13 (dd, 3 J = 3.2 Hz, 1H, CH). 13 C 1.10 -3 g.mL -1 , EtOH) = -16.0°. (dd, J = 4.4 Hz and 1.6 Hz, 2H, HAr), 7.86 (dd, J = 4.4 Hz and 1.6 Hz, 2H, HAr), 3.94 (s, 3H, 297.895 g mol -1 ) C, 24.03; H, 2.69; N, 9.34. Found: C, 24.74; H, 3.19; N, 9.65 %. [α]D (c = dark. White crystals were obtained. M.p. 134°C. 1 H NMR (300 MHz, acetonitrile-d3) δ 8.79 (CH), 154.89 (CONBr), 169.36 (NBrCONBr). Elem. Anal. Calcd. for C6H8Br2N2O2 (M = method. Hexane was used as precipitant. The sample was left at 2°C during 8 days in the NMR (ACN-d3, 75.5 MHz) δ 15.82 (CH(CH3)2), 16.45 (CH(CH3)2), 29.52 (CH(CH3)2), 72.68 A c c e p t e d 10 -5 mol) were dissolved in dichloromethane (4 mL). The solution was filtered to remove non-dissolved particles. The solution was left to evaporate during 8 days in the dark at room temperature. White crystals were obtained. M. p. 196°C. 1 H NMR (300 MHz, DMSO-d6) δ 8.96 -8.79 (m, 2H), 7.79 (m, 2H), 1.31 -0.97 (m, 6H). Elem. Anal. Calcd. for C11H10Br2F3N3O2 (M = 433.02 g mol -1 ): C, 30.51; H, 2.33; N, 9.70 %. Found: C, 30.50; H, 2.64; N, 11.18 %. DBH•[Py-CO2Me]. DBH (15 mg, 5.25 10 -5 mol) was dissolved in chlorobenzene (2 mL). 4-Methylisonicotinate (21 µL, 15.75 10 -5 mol) was added. The solution was filtered to remove non-dissolved particles and poured in a Durham tube. Crystals were made by vapor diffusion A c c e p t e d g.mL -1 , EtOH) = + 66.1°. m a n u s c r i p t Synthesis of the adducts. DBH•[Py-CN]. DBH (15 mg, 5.25 10 -5 mol) was dissolved in 1,2-dichloroethane (2 mL). 4-Cyanopyridine (15 mg, 15.75 10 -5 mol) was added. The solution was filtered to remove non-dissolved particles and poured in a Durham tube. Crystals were obtained by vapor diffusion method. Hexane was used as precipitant. The sample was left to 2°C during 8 days in the dark. White crystals were obtained. M. p. 148°C. 1 H NMR (300 MHz, DMSO-d6) δ 8.81 (dd, J = 1.6 and 4.5 Hz, 2H, HAr), 7.67 (d, J = 1.6 and 4.5 Hz, 2H, HAr), 1.39 (s, 6H, CH3). Elem. Anal. Calcd for C11H10Br2N4O2 (M = 387.917 g mol -1 ): C, 33.87; H, 2.58; N, 14.36 %. Found: C, 33.99; H, 2.44; N, 14.37 %. DBH•[Py-CF3]. DBH (15 mg, 5.25 10 -5 mol) and 4-trifluoromethylpyridine (30 µL, 15.75 crystals were obtained. M. p. 159°C. 1 H NMR (300 MHz, DMSO-d6) δ 8.66 (s, 4H, HAr), 1.50 (s, 12H, CH3). Elem. Anal. Calcd. for C14H16Br4N6O4 (M = 651.936 g mol -1 ): C, 25.79; H, 2.47; N, 12.89 %. Found: C, 25.97; H, 2.44; N, 12.71 %. [1-iBu]•[Py-CO2Me]. 1-iBu (15 mg, 4.78 10 -5 mol) was dissolved in 1,2-dichloroethane (2 a n u s c r i p t mL). 4-C14H17Br2N3O4 (M = 451.115 g mol -1 ): C, 37.28; H, 3.80; N, 9.31 %. Found: C, 37.80; H, m 3.86; N, 9.44 %.

Table 2

 2 Crystallographic data

		(DBH)2•	DBH•[Py-CN]•	DBH•	DBH•	[1-iBu]•
		Pyrazine	(C2H4Cl2)0.5	[Py-CF3]	[Py-CO2Me]	[Py-CO2Me]
	Formula	C7H8Br2N3O2	C12H12Br2ClN4O2 C11H10Br2F3N3O2 C12H13Br2N3O4	C14H17Br2N3O4
	FW (g.mol -1 )	325.98	439.53	433.04	423.07	451.12
	System	triclinic	monoclinic	monoclinic	triclinic	orthorhombic
	Space group	P-1	C2/m	P21/c	P-1	P212121
	a (Å)	7.7140(4)	8.6301(5)	9.4108(4)	6.1692(8)	6.5779(5)
	b (Å) c (Å) á (deg) â (deg) ã (deg) V (Å 3 ) T (K) Z Dcalc (g.cm -1 ) µ (mm -1 ) Total refls Abs corr Tmin, Tmax θmax (°)	8.0925(4) 9.1626(4) 64.045(3) 81.550(4) 85.532(4) 508.65(4) 150(2) 2 2.128 7.946 3773 multi-scan 0.106, 0.356 25.03	7.7579(7) 23.769(2) 90.00 98.599(4) 90.00 1573.5(2) 150(2) 2 1.855 5.330 4030 multi-scan 0.209, 0.619 27.41 m 7.9826(3) 19.9558(10) 90.00 97.418(2) 90.00 1486.59(11) 150(2) 4 1.935 5.492 8226 multi-scan 0.429, 0.547 27.52 a n u s c r i p t 7.9700(10) 7.9462(5) 15.743(2) 32.486(2) 88.236(5) 90.00 81.458(5) 90.00 85.411(5) 90.00 762.90(17) 1698.0(2) 150(2) 150(2) 2 4 1.842 1.765 5.331 4.796 5692 13213 multi-scan multi-scan 0.116, 0.726 0.587, 0.746 27.58 27.473
	Uniq refls Rint Uniq refls [I > 2σ(I)] R1 wR2 (all data) Flack param. GOF Res. dens. (e Å -3 ) A c c e p t e d 1799 1929 0.0308 0.0417 1539 1609 0.0282 0.0536 0.0775 0.1428 --1.108 1.058 0.482, -0.746 1.603, -1.841	3382 0.0378 2684 0.0339 0.1177 -0.819 0.650, -0.801	3446 0.0368 2684 0.0559 0.1774 -1.035 1.422, -1.173	3881 0.0503 3351 0.0313 0.0574 0.011(8) 1.048 0.405, -0.420

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