Synthesis of New Trimeric Lignin Model Compounds Containing 5-5 ’ and β-O-4 ’ Substructures , and their Characterization by 1 D and 2 D NMR Techniques

Os trímeros-modelo de lignina contendo as subestruturas bifenila (5-5’) e aril-éter (β-O-4’) foram sintetizados a partir dos derivados da desidrodivanilina e da α-bromo acetovanilona pela reação de Williamson. O estudo de RMN de 1H e de 13C destes trímeros foi feito utilizando as técnicas homo e heteronucleares. A atribuição dos sinais de RMN de 1H e de 13C e a conformação das moléculas são também discutidas neste artigo.


Introduction
Lignin is an integral component of wood consisting approximately one-third of the woody material in vascular plants.In general, lignin is removed in wood based industries, particularly in pulping processes in the pulp and paper industry, as by-products, usually burned in the recovery furnace to produce energy for mill operation.However, there is an increasing interest in its structural study because of its potential utilization as chemical feedstock in the chemical industry 1,2 .
The objectives of this series of investigation are, therefore three-fold: (a) to synthesize trimeric lignin model compounds, (b) to study 1 H and 13 C NMR spectroscopic characteristics of this class of compounds, and finally (c) to compare these NMR spectroscopic characteristics to clarify the presence of these substructures in lignin.
Compound I was prepared from 4-O-acetyldehydrodivanillin and α-bromo-3-methoxy-4-ethoxyacetophenone via Williamson's reaction.Compounds II, III, and IV were obtained from I by base-catalyzed hydrolysis, hydroxymethylation with paraformaldehyde via aldol addition, and reduction, respectively.For the NMR studies DEPT, PEN-DANT, nOe difference, and 2D HMBC, 1 H/ 1 H-COSY and 1 H/ 13 C-COSY techniques were used to obtain the 1 H and 13 C NMR spectroscopic characteristics of the compounds.

4'-O-[α-(3-methoxy-4-ethoxyphenyl)-α-oxo-β-hydroxymethylethyl] dehydrodivanillin (III)
A solution of I (0.10 g, 0.18 mmol), paraformaldehyde (0.026 g, 0.3 mmol) and NaOH (0.0076 g) in 5.5 mL of DMF was stirred on an oil bath at 60 o C under a N 2 atmosphere for 12 h.The reaction mixture was then poured into 10 mL of ice-cooled water.The precipitate was filtered off, and the aqueous layer was extracted with CHCl 3 , and the solvent was removed under reduced pressure.The product was precipitated by addition of ice-cooled water into the resulting solution to give a beige solid (0.010g, 10%

Results and Discussion
The chemical shift assignments and coupling constants for I and II (Table 1) were deduced from 1D NMR spectra, and confirmed by 1 H/ 1 H-COSY, HMBC and nOe difference spectra.For example, for compound I: the 1 H resonances at δ 7.33 (doublet, J 1.93Hz), 6.97 (doublet, J 8.45Hz) and 7.47 (double doublet J 1.93 and 8.45 Hz) indicate the three spin system of ring C and were confirmed by HMBC technique (Table 2) and nOe difference spectra (Table 3).
The HMBC data confirmed the aromatic and formyl hydrogen chemical shifts for rings A and B. Hence, Table 2 data show that methylene at δ 5.34 (β to B and C) as well as both H-2 at δ 7.60 and H-6 at δ 7.38 are correlated with C-4 at δ 152.6.These correlations confirm ring B hydrogen assignments.H-2 at δ 7.60 also correlates with formyl carbon at δ 192.4 and C-6 at δ 126.3.Once the hydrogen chemical shifts for rings B and C were assigned, the remaining ones were attributed to ring A. Finally, H-2 at δ 7.57 shows correlation with formyl group of ring A at δ 192.7.
The nOe difference spectra are useful in establishing molecular conformation and steric arrangement of substituents.Therefore, spectra obtained by using these techniques were used to verify the conformation of I (Table 3).Irradiation of OCH 2 CO hydrogens at δ 5.34 enhances the signals of H-2" at δ 7.33 and H-6" at δ 7.47, both aromatic hydrogens in the ring C of compound I, while the intensity of the signal at δ 3.82, corresponding to hydrogens of methoxy group in ring B, was not affected.This last observation shows that the C 4' -O bond is not free to rotate.On the other hand, irradiation of the methoxyl group resonance at δ 3.82 only enhances the signal at δ 7.60 corresponding to H-2' of ring B.
These results show that the 3-methoxy-4-ethoxyphenacyl group is syn to ring A and also that ring C is rotating about C α -C 1'' bond.In addition, the analysis of nOe difference spectra has allowed to assign unambiguously the chemical shifts of hydrogens and methoxy groups of each aromatic ring (Table 3).
By combining the data obtained from 13 C NMR, DEPT, and PENDANT spectra with those of 2D 1 H/ 13 C -COSY contour plot it was possible to assign the aromatic hydrogenated carbons for each A, B, and C rings of trimers I and II. 1 H/ 13 C -COSY data are summarized in Table 4.
1 H NMR spectra of trimers III and IV were analyzed using the 1 H NMR spectra of both compounds I and II as a reference.This leads to assignments of hydrogens in trimers III and IV.The results are summarized in Table 5.
In addition, the presence of a hydroxymethyl group in trimer III was verified by comparing chemical shifts of trimer II (Table 1) with those of trimer III (Table 5).This group was characterized by signals at δ 4.87 (CH 2 OH) and δ 4.81 (CH 2 OH).As for trimers I and II, the 13 C chemical shift assignments for compounds III and IV were deduced from 13 C NMR spectra with help of the corresponding DEPT spectra and 1 H/ 13 C -COSY.
The 13 C spectrum of trimer III exhibited signals for aliphatic carbons at δ 14.6 (CH 3 CH 2 ), 55.3, 55.7, and 55.9 (OCH 3 ), 64.0 (CH 3 CH 2 O), 62.5 (OCHCH 2 OH), 83.8  6 summarizes the 13 C NMR chemical shifts of aromatic carbons in trimers I to IV.The chemical shifts of aromatic carbons for compound III were assigned by comparison with those of compound II, while those of aromatic carbons in trimer IV were determined by comparison with model compounds 1 and 2 (Figure 2) 10,11 .The chemical shift assignments for compoud IV were also deduced from the 13 C NMR spectrum with help of the corresponding DEPT spectra.
Table 7 shows the 13 C NMR chemical shifts for these three compounds.From these data it may be verified that aromatic carbons of rings A and B of trimer IV as well as those of parent compound 1 present very close chemical shifts.Similarly, the chemical shifts assigned to aromatic carbons of ring C are closely related to those of compound 2.
The chemical shifts and coupling constants for I and II were deduced from 1D NMR spectra, and confirmed by HMBC, 1 H/ 1 H-COSY and nOe difference spectra.
This last one was useful in establishing the conformation of 4-O-acetyl-4'-O-[α-(3-methoxy-4-ethoxyphenyl)-  show that the 3-methoxy-4-ethoxyphenacyl group is syn to ring A and also that ring C is rotating about C α -C 1'' bond with no restriction.In addition, the analysis of nOe difference spectra has allowed to assign unambiguously the chemical shifts of hydrogens and methoxyl groups of each aromatic ring. 1 H NMR spectra of trimers III and IV were analyzed using the 1 H NMR spectra of both compounds I and II as a reference.As for trimers I and II, the 13 C chemical shift assignments for compouds III and IV were deduced from 13 C NMR spectra with help of the corresponding DEPT, 1 H/ 13 C COSY and HMBC spectra.While the chemical shifts of aromatic carbons for compound III were assigned by comparison with those of compound II, those of aromatic carbons in trimer IV were determined by comparison with dimeric model compounds.

Furthermore, chemical structures
of trimers II and IV, differ in the substituents at C-1 of rings A/B and at C-α of side chain in ring C. As compared to trimer II, C-4 and C-6 of ring A of trimer IV are shielded by ∆δ -5.6 to -7.0, while C-1 and C-5 are deshielded by ∆δ + 4.4 and + 1.1, respectively.For ring B of IV, C-2 and C-4 are shielded by ∆δ -1.2 and -5.8, respectively, in addition to deshielding of C-1, C-3 and C-5 (∆δ + 1.6-5.7).For ring C of IV, C-4 and C-6 undergo shielding of ∆δ -5.0 and -3.9, respectively, and C-1 deshielding of ∆δ + 3.8.

II III IV - Figure 1. Synthetic route for trimers I to IV.
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Table 1 .
1H Chemical Shifts (δ) of trimers I and II

Table 2 .
H/C correlations ( 3 J CH ) for model compound I using

Table 3 .
Interpretation of nOe difference spectra* of trimer I