A recently described plant cell wall dissolution system has been modified to use perdeuterated solvents to allow direct in-NMR-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent system containing dimethylsulfoxide-d 6 and 1-methylimidazole-d 6 in a ratio of 4 : 1 (v/v), keeping wood component structures mainly intact in their near-native state. Two-dimensional NMR experiments, using gradient-HSQC (heteronuclear single quantum coherence) 1-bond 13 C-1 H correlation spectroscopy, on nonderivatized cell wall material from a representative gymnosperm Pinus taeda (loblolly pine), an angiosperm Populus tremuloides (quaking aspen), and a herbaceous plant Hibiscus cannabinus (kenaf) demonstrate the efficacy of the system. We describe a method to synthesize 1-methylimidazole-d 6 with a high degree of perdeuteration, thus allowing cell wall dissolution and NMR characterization of nonderivatized plant cell wall structures.

We have shown that the reaction of guanosine with chloroacetaldehyde in aqueous solution in the physiological pH range yields 1,N2-ethenoguanosine (5,9-dihydro-9-oxo-3-~-~-ribofuranosylimidazo[l,2-a]purine). This compound could be hydrolyzed to 1,N2-ethenoguanine (5,9-dihydro-9-oxoimidazo[l,2-a]purine), which was also prepared authentically by hydriodic acid treatment of the glyoxal-guanine adduct. The 1,N2-ethenoguanine, which is an unsubstituted (at N-4, C-6, and C-7) Y-type base, is not fluorescent under the same conditions at which the 4-methyl compounds fluoresce. By contrast, the isomeric and angular N2,3-ethenoguanine (8,9-dihydro-9-oxoimidazo(2,l-blpurine) is fluorescent (bmitation 262 nm, Xemjmion 410 nm). The N2,3-ethenoguanine synthesis was initiated by the reaction of chloroacetaldehyde with 06-benzylguanine, 06-methylguanine, and 2-amino-6-benzylthiopurine, followed by hydrogenolysis or hydrolysis, hydrolysis, and oxidation and hydrolysis, respectively. The reaction of guanosine is indicative of the damage that can result from the action of the mutagen chloroacetaldehyde on guanosine derivatives under physiological pH conditions. The reaction of chloroacetaldehyde in aqueous solution with adenine-and cytosine-containing compounds1p2 to produce 1a6-and 3,N4-etheno-bridged compounds, respectively, has found wide a p p l i~a t i o n .~~~ Interest stems from the biological activity generally evident at the nucleoside, nucleotide, and coenzyme level and from the species responsible for the fluorescence emission properties.596 The crystal and molecular structures of suitable derivatives have been We agree with Kochetkov, Shibaev, and Kostl that in the pH range most favorable for reaction at 37 OC of chloroacetaldehyde with adenosine (pH 4.5) and cytidine (pH 3.5), guanosine is not reactive.2 When chloroacetaldehyde was used in this laboratory to modify tRNA in aqueous solution at different selected ~H S ,~ guanosine as well as cytidine and adenosine residues appeared to be undergoing attack at pH 6.3,lO within the optimum range for retention of tRNA tertiary structure.Since chloroacetaldehyde is known to be mutagenic12.13 and is one of the likely liver metabolites of vinyl chloride, its reaction with guanosine under physiological conditions was of particular interest. Moreover, the possible development of fluorescence due to the formation of an additional ring suggested the value of product comparison with the fluorescent natural nucleosides Y1p27 (wybutosine, Y -W Y O )~~ and YtZs3l (wyosine, W Y O )~~ and corresponding bases related to guanine.32The reaction of guanosine (1) with chloroacetaldehyde in aqueous solution at 37 "C was followed by the development of ultraviolet absorption at 305 nm over a period of hours and over a pH range from 6.5 to 4.5. At pH 6.5 the reaction rate is significant, but is still less than one-third that of adenosine with chloroacetaldehyde under the same conditions. The relative reaction rate for the guanosine reaction falls off sharply with decreasing pH and is practically ...

The standard Oliver-Pharr nanoindentation analysis tacitly assumes that the specimen is structurally rigid and that it is both semi-infinite and homogeneous. Many specimens violate these assumptions. We show that when the specimen flexes or possesses heterogeneities, such as free edges or interfaces between regions of different properties, artifacts arise in the standard analysis that affect the measurement of hardness and modulus. The origin of these artifacts is a structural compliance (C s ), which adds to the machine compliance (C m ), but unlike the latter, C s can vary as a function of position within the specimen. We have developed an experimental approach to isolate and remove C s . The utility of the method is demonstrated using specimens including (i) a silicon beam, which flexes because it is supported only at the ends, (ii) sites near the free edge of a fused silica calibration standard, (iii) the tracheid walls in unembedded loblolly pine (Pinus taeda), and (iv) the polypropylene matrix in a polypropylene-wood composite.

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