Direct Identification of Diverse Alicyclic Terpenoids in Suwannee

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Direct Identification of Diverse Alicyclic Terpenoids in Suwannee River Fulvic Acid Neal Arakawa, and Lihini I. Aluwihare Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5055176 • Publication Date (Web): 13 Mar 2015 Downloaded from http://pubs.acs.org on March 25, 2015

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Environmental Science & Technology

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Direct Identification of Diverse Alicyclic Terpenoids

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in Suwannee River Fulvic Acid

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Neal Arakawa*. Lihini Aluwihare

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Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Drive, La

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Jolla, CA 92093-0244.

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The chemical complexity of natural dissolved organic matter (NOMDOM) obstructs our ability

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to definitively recover source compounds from within NOMDOM, an objective which has the

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capacity to alter our understanding of carbon sequestration on a global scale. To advance

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compositional studies of NOM DOM we have applied a previously published reduction method

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to an environmental standard, Suwannee River Fulvic Acid (SRFA). The reduction products,

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comprising 12% of the pre-reduced carbon, were then separated by comprehensive two-

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dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOF-MS). Results

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indicate that the majority of observed reduced compounds corresponded to alicyclic

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hydrocarbons in the size range C10 to C17. Cyclic terpenoids are the only biomolecule class with

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contiguous, alicyclic carbon backbones of this size. These terpenoid reduction products contain

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series offset by CH2 and exhibit great isomeric diversity, features previously inferred from

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ultrahigh resolution mass spectrometry and NMR studies of unreduced SRFA. Reduction of

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Taxoddinium leaf litter as a source material to SRFA confirmed the prevalence of terpenoids in

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SRFA and provided insight into the parent compounds that must be diagenetically modified on

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ACS Paragon Plus Environment


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relatively short timescales. These data corroborate several recent studies that suggest alicyclic

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hydrocarbons to be important components of longer-lived DNOM.

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Abstract Art

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Introduction

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Dissolved organic carbon (DOC) from aquatic environments is most easily divided into two

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classes of material; compounds of identifiable structure and biochemical origin (e.g., lignin

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phenols, sugars, lipids), and compounds with unknown structure (e.g., typically termed humic

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substances [humic acid, fulvic acid, humin], protokerogen). The majority of Dissolved organic

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matter (DOM) represents DOC falls into the latter group, which makes it a quantitatively

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significant reservoir of the global carbon cycle. 1, 2 Yet, its provenance remains elusive, as much

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of it has been immune to degradative compositional interrogation. Recent studies employing

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nuclear magnetic resonance spectroscopy3-5 (NMR) and Fourier transform ion cyclotron

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resonance spectrometry6,7 (FT-ICR-MS) have implicated linear and alicyclic terpenoids as major

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precursors of terrestrial and marine DOM. Sources of terpenoids and their biosynthetic pathways

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are well known, and so, direct identification of the relevant structures and their diversity could

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hold significant promise for linking DOM composition to source.8

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Formatted: Indent: First line: 0"

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Some of the earliest reports of terpenoid precursors in DOM are from studies of Suwannee River

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Fulvic Acid (SRFA), a widely used environmental DOM standard. For this reason, numerous

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attempts have been made to compositionally deconstruct humic substances (used loosely here to

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refer to DOM that is poorly characterized). For such studies, Suwannee River Fulvic Acid

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(SRFA) has been heavily used as an environmental natural organic matter (NOM) standard in the

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development of analytical techniques.

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The parent material of SRFA is organic matter leached from leaves or soil.3 However,

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biological and abiotic processes have transformed these compounds into an extremely

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chemically diverse mixture with a broad molecular size range. Humic substances and SRFA

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specifically, have received decades of attention.4,

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application of ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry

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(FT-ICR-MS) to detect thousands of compounds, many of which are assigned a specific

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elemental formula.6, 7 In addition, 13C and multi-dimensional NMR techniques have been used to

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provide a broad compositional context for SRFA including the identification of individual

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chemical structures. The sum of the body of current work indicates that SRFA is derived from a

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mixture of lignin, tannin, and terpenoid (including sterol) precursors. 8-113-5, 9 Yet, quantitatively

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little lignin, and no definitive tannin or terpenoid structures, have been isolated from humic

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substances12 substances10

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More recent studies have focused on the

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The recovery of specific structures from SRFA and other natural organic matter (NOM) would

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not only confirm source material identity, but also has the potential to inform our understanding

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of the processes that alter DOM on the molecular level. As with previous studies we use SRFA

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as a model for DOM and test a method that is specifically designed to access hydrocarbon

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backbone structures.

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Although fractionation of SRFA via liquid chromatography is improving, isolation of

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individual compounds is currently unattainable.13

11

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separating capabilitieson, but much of SRFA is likely too large and polar to be analyzed

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natively.6, 7 However, chemical degradation, such as chemical reduction, is a viable method for

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making SRFA more GC-amenable. Previous studies employing an n-Butylsilane reduction

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method reported the separation of various products from SRFA, but much of the mixture was left

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unresolved.14, 1512,13 In this study, the reduction method has been adapted and combined with

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comprehensive gas chromatography (GCxGC) coupled to time-of-flight mass spectrometry

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(TOF-MS), to provide additional molecular-level data on the composition of SRFA.

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GCxGC technique uses an additional column to provide unsurpassed chromatographic

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resolution, especially when applied to hydrocarbons.16

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presence of cyclic terpenoid derived carbon backbones compounds in SRFA, and demonstrates

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both the unique capability of this approach and its potential for further use.. The unique

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capability of this approach complements the existing suite of degradation techniques and can

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provide a more comprehensive molecular level understanding of DOM source and fate.

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Materials and Methods

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Materials. All solvents (ACS grade or better), model compounds and hydrocarbon standards

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were purchased commercially, and used as supplied. Suwannee River Fulvic Acid (2S101F) was

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purchased from the International Humic Substances Society.

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Tris(pentafluorophenyl)borane (B(C6F5)3) were purchased from Sigma Aldrich, and stored in an

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Argon filled dessiccator when not in use. Dichloromethane (CH2Cl2) was stored over activated

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molecular sieves (3 Å, 10% wv). Freshly fallen Lleaf litter (based on intactness of leaves) was

Gas chromatography (GC) offers better

14

This

Data presented here confirms the

n-Butylsilane (n-BS) and

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collected from Tifton, GA, and verified to be of the genus Taxodinium. Leaves were freeze-

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dried for 1 week, and ground using a mortar and pestle before reduction.

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Reduction Procedure. The reduction procedure was modeled after previous studies.14

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model compound reductions, compounds (~1 mg of each) were weighed (balance accurate to 0.1

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mg) into a single, flame dried 2 mL vial equipped with a stir bar. Under argon atmosphere, 10

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mg. of B(C6F5)3 was added (100 μL of 100 mg B(C6F5)3/1 mL CH2Cl2 solution). Immediately

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after addition, 100 μL of n-BS was added, also under argon. The mixture was then allowed to

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stir-overnight. Following reduction, the samples were treated by the following procedure to

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remove excess catalyst and siloxane by-products. Subsequently 50 μL of the pot was syringed

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into 250 μL of conc. potassium hydroxide in methanol (KOH/MeOH). After 1 minute, the

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mixture was extracted with pentane (3x100 µuL). The pentane fractions were collected, washed

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with H2O, and dried over sodium sulfate. The organic fraction was then dried under N2 to 100

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μL for GC-FID/MS and GCxGC-TOF-MS analysis. The same procedure was used for the

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reduction of SRFA and leaf litter, with 10 mg. of starting material for each. All reductions were

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carried out in duplicate. While SRFA was reduced to dissolution, particulates were still observed

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in the leaf litter reduction were still observed.

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For

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Reaction conditions were selected based on results from model compound reductions. The

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amount of B(C6F5)3 used was found to be the most critical parameter affecting the reduction

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yield of model compounds.

Chart 1 lists model compounds and their observed reduction

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products. In a previous work with reduction of 1a (Chart 1, 10 mg), which was the most

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chemically resistant of the model compounds tested, it was observed that increasing the amount

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of B(C6F5)3 to 10 mg also increased reduction yields, whereas increasing n-BS did not. The

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ratio of 10 mg B(C6F5)3 to 10 mg (or ~5 mg organic carbon) starting material was therefore

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selected to ensure complete reduction of oxygen containing groups in model compounds as well

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as in environmental samples.

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B(C6F5)3 addition, as environmental samples are extremely polydisperse and therefore make the

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use of molar equivalents impractical. This quantity of catalyst gave the most predictable and

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consistent results across all model compounds and SRFA.

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Gas Chromatography-Flame Ionization Detection/Mass Spectrometry (GC-MS/FID). An

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Agilent 7890A gas chromatograph coupled to an Agilent 5975C quadrupole mass spectrometer

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was used for chromatographic separation and compounds were detected simultaneously with

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Electron Ionization MS and FID, enabled by a splitter. Splitless injection with 1 μL of analyte

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was performed onto a 5% phenyl poly(dimethylsiloxane) column (J&W 123-5731DB-ht, 30 m,

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320 μm i.d., 0.1 μm film) and separated using a temperature program from 50 °C ( hold time 0.5

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min) to 140 °C (at 10 °C/min, hold time 0 min) to 320 °C (at 15 °C/min, hold time 10 min).

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Helium was the carrier gas with a constant flow of 1.8 mL/min, the FID was operated at 310°C

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and the mass spectrometer conditions included 70 eV ionization with scanning from 50-750 m/z.

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The transfer line was set at 280 °C. Quantification of reduced model compounds and reduced

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SRFA was accomplished using a hydrocarbon standard (decahydronapthalene) and assuming a

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constant FID response factor across hydrocarbons. When available, reduced model compound

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structures were identifiedconfirmed by comparison to the NIST Mass Spectral Standard

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Reference Database (2005). For SRFA, manual integration was used to remove peaks associated

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with catalyst degradation and residual siloxanes, and sample MS are located in Supporting

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Information.

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Comprehensive Two-Dimensional Gas Chromatography (GCxGC-TOF-MS).

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Pegasus 4D GCxGC-Time-of-Flight Mass Spectrometer (GCxGC-TOF-MS) was used for

Quantity is the most practical measure for both n-BS and

A LECO

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comprehensive GC analysis.15 The term GCxGC refers to the use of two orthogonal columns,

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that is, each column has a different chemical selectivity which were selected to increase the

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separation of aliphatic from aromatic hydrocarbons. Compounds are separated primarily by

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volatility in the first column and polarity in the second. The analysis is comprehensive because

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all of the effluent from the first column is cryofocused and transferred onto the second column.

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Effluent from the second column is then analyzed by the TOF-MS, which benefits from high

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spectral acquisition speed (50 Hz-500 Hz). Finally, the data is compiled into a two-dimensional

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chromatogram that is visualized and processed by ChromaTOF® software. Both columns are

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housed within an Agilent 7890A gas chromatograph. The splitless inlet temperature was set at

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300 °C.

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polysiloxane column (Restek Rxi-17Si, 30 m length, 0.2550mm i.d., 0.25 μm film thickness).

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The column was programmed to remain isothermal at 40°C for 1 min, and ramped to 315 °C at 3

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°C per minute. The modulator temperature was offset by +30 °C to the primary oven. The

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secondary oven (within the GC) housed the second dimension non polar Crossbond® dimethyl

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polysiloxane column (Restek Rxi-1, 1.58m length, 0.250mm i.d., 0.25μm film thickness). The

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secondary oven temperature was offset by +25°C to the primary oven. The modulation period

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was 2.5 s, with a hot pulse time of 1.05 s and a cool time of 0.2 s. The carrier gas was Helium at

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a constant flow of 1.5 mL/min. The acquisition delay on the TOF-MS was set to 160 s. The

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acquisition rate was set to 50 Hz, with a range of 5-1000 m/z. The transfer line was set at 280

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°C. Electron Ionization Impact was run at 70 eV. Again, reduced model compound structures

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were confirmed identified by comparison to the NIST Mass Spectral Standard Reference

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Database (2011).

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Results and Discussion

The first dimension column was a semi-polar Crossbond® diphenyl dimethyl

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Model Compound Analysis. The reduction process was applied to a collection of chemically

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distinct model compounds with functional groups that are expected to also be present in SRFA.

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This was done to verify that the products were both consistent with previously published

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literature and consistently reproducible. We observed that the catalyst (B(C 6F5)3) and by-product

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siloxane polymers often coelute in high concentrations with analytes of interest, impacting

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accurate GC-FID quantification and complicating GCxGC analysis.

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KOH/MeOH to remove these by products, which resulted in cleaner chromatograms. However,

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this method also removes reduction products which are polysilylated, such as phenols (the

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reduction products of lignins and tannins) and some hydrosilylated (e.g. 2g, 2h, Chart 1)

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compounds.

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the topic of this study. Below, we systematically describe the effect of chemical reduction on

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different functional groups, as supported by selected substrates and their reduced products.

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Chart 1. Reduction of model compounds with n-butylsilane/ B(C6F5)3 Entry

We used concentrated

Recovery of phenols can be achieved by acid hydrolysis of silyl ethers, but are not

Substrate (1)

Product(s) (2)

GC Yield

1

58%

2

78%

3

65%

4

65%

5

72%

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No observed product

N.A.

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*25%

8

*25%

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51%

10

34%

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*Products 2g and 2h coelute. GC yield calculated as average

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The reduction process was adapted from previous work,14 , 1512,13 and the reaction mechanism is

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reproduced in Supporting Information. All of the carboxylic acids in model compounds (1a, 1e,

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1i, and 1j) reduced completely to methyl groups, as predicted. The structure of 1a is likely to

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rival the most sterically-hindered arrangement of oxygen containing functional groups in SRFA,

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and is potentially a good chemical proxy. Ketones such as in 1b are labile, reducing completely

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to methylene groups. Aldehydes such as in 1c are also labile, reducing completely to methyl

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groups. Ester linkages, such as in 1d, cleaved and reduced completely. This product was not

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expected based on previous studies14

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preservation of the newly created ether linkage was observed. Aliphatic alcohols are reduced,

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but as is shown in 1e they can also be eliminated in particular cases, resulting in a double bond.

studies12

in which reduction of the carbonyl but

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In contrast to previous studies, we have found that with a few exceptions double bonds are

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labile, and this can be attributed to the higher catalyst concentrations employed here. In the

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reduction of linear diene 1f no products are observed. This is believed to be the result of a

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hydrosilylation reaction at the double bond(s).

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reacted with reducing agent by-products, forming compounds which are lost during the post-

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reduction workup. This behavior is observed in branched diene 1g and cyclic alkene 1h. The

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hydrosilylation products 2g and 2h are in fact isolated but in low yields, which indicates that

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material is lost to further reactions. Hydrosilylation of double bonds has been observed in

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previous literature under similar conditions.17

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easily identified by distinctive fragmentation patterns in mass spectra.

16

The resulting organosilane product further

The incorporation of n-BS into products is

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In addition to hydrosilylation, double bonds can also be reduced under reaction conditions. In

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the reduction of 1i, the isolated product 2i is fully reduced. However, the reduction product 2j is

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only partially reduced. This is because double bonds present in a ring system are more resistant

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to reduction, as is observed for products 2c and 2e. That double bonds are most stable between

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tertiary carbons is clearly seen in the difference between the reduction products 2c and 2h, where

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the double bond is preserved in 2c but hydrosilylated in 2h. The mechanism by which double

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bonds are reduced vs. hydrosilylated is not yet fully understood. There does appear to be a

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correlation of reduction with conjugated compounds (1i, 1j), however in a later section we

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observe that non-conjugated terpenes are also reduced.

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The reduction of 1e and 1j produces multiple isomers of products with identical molecular ions

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and similar fragmentation patterns. Previous studies have shown that Lewis acid catalysts enable

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backbone rearrangement (double bond migration and methyl shifts) in unsaturated sterol

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hydrocarbons.1178 In those studies and our current work, rearrangements give rise to multiple

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isomers from the reduction of a single compound, and the relative amounts of these products can

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be altered by the amount of catalyst used. A combination of GC-MS, 13C NMR, and 1H NMR of

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the reduction products was used to determine the location of the double bond in 2e. A detailed

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description of this derivation is provided in Supporting Information. Thus far, isomer production

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has only been observed in fused cyclic structures, which contained either double bonds or

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hydroxyl groups directly bonded to the ring system prior to reduction. It is further proposed,

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though not tested here, that other functional groups incorporated into the ring backbone, such as

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ketones, would also behave in a similar manner to the hydroxyl reduction.

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Finally, we have observed that the reduction of 1e produces both fully (2e’) and partially

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reduced products (2e), with a molecular ion offset of 2 m/z. It appears that the partially reduced

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products are not intermediates to the fully reduced product 2e’, as an increase in catalyst only

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results in the loss of 2e (likely due to hydrosilylation) without an increase in 2e’. As such, it

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appears that both are produced simultaneously by different pathwaysdifferent pathways generate

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both reduction products simultaneously.

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behavior. The production of multiple reduction products with different numbers of double bonds

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from the same reactant ultimately prevents assignment of unsaturation state to a reactant of

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unknown composition.

Of the compounds tested, only 1e exhibited this

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In summary, it was found that all oxygen-containing groups, with the exception of phenols, are

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reduced, that aromatic double bonds are stable but that aliphatic double bonds are only preserved

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in ring systems, and that carbon-carbon bonds are not made or broken. The more complete

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reduction compared to previous work12,13 is attributed to the elevated catalyst concentration that

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is employed in the current method.

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standards and samples.

Excess catalyst has been consistently used across all

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Comprehensive GCxGC-TOF-MS. The distribution of hydrocarbon standards and reduced

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model compounds in GCxGC space (Figure 1) mapped a chemical landscape that enables

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preliminary assignment of broad functionality to unknown hydrocarbons based on 1st and 2nd

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dimension retention times (1RT and 2RT respectively). Colored areas indicate where saturated

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(alkane), unsaturated, and aromatic hydrocarbons are expected to elute. A thorough description

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of the assignment of these regions is provided as Supporting Information.

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Figure 1. A GCxGC selected-ion chromatogram (71 m/z) identifying the elution geography of

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n-alkane standards. Circled regions overlay areas where depicted reduced model compounds and

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hydrocarbons elute. Boxed compounds are isomers of aromatic C12H18 compounds. Note that

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the reduction products of 1e and 1j elute as a series of peaks over a large region.

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Terpenoids in SRFA: As previously mentioned, we were unable to separate the UCM into

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discrete peaks using GC-FID/MS. However, GC-MS data still proved to be useful for structural

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characterization. In a time averaged, baseline subtracted spectrum (Figure 2b) taken along the

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UCM we observe clusters of prominent fragmentation ions which are spaced 14 m/z apart

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(Figure 2c). Based on previous knowledge of EI-MS hydrocarbon fragmentation we suspected

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that many closely related compounds contributed these fragments and used this knowledge to

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further interrogate the sample by GCxGC-TOF-MS.

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Three series of compounds are largely responsible for the diversity of fragment ions observed

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in reduced SRFA by GCxGC-TOF-MS (Figures 2b, 2c). Each series contains many individual

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compounds which occupy a two dimensional GCxGC space. These three series are displayed in

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Figures 3a, 4a and 5a in 97 m/z, 95 m/z, and 107 m/z single ion chromatograms. Within each

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series individual compounds yield very similar mass spectra, as certain fragment ions are highly

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conserved across the series. For example, in the hydrocarbon series identified with 97 m/z

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(Figure 3a) we observe 3 prominent fragmentation ions: 83 m/z, 97 m/z and 111 m/z (Figure 3c).

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These ions are spaced at 14 m/z, the same spacing observed in GC-MS of the UCM, and are

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present in almost all observed peaks from this series. Compounds identified in Figure 4a have

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prominent fragment ions of 81 m/z, 95 m/z and 109 m/z (Figure 4c), and compounds in Figure

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5a have ions of 79 m/z, 93 m/z and 107 m/z (Figure 5c). All together, these three series

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represent the prominent fragment ions observed in GC-MS analysis. Additionally, the large

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molecular size range (1RT range) and abundance distribution of each series are similar to the

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features observed in the GC-FID chromatogram (Fig. 2a), and confirms the GCxGC observation

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that the UCM is largely represented by the three series of compounds presented.

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Figure 2-5. GC-FID chromatogram (2a) of reduced SRFA. GC-MS mass spectrum (2b) of

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material eluting from 10.0-17.6 minutes.

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fragmentation ions.

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Selected- ion chromatograms of 97 m/z (3a), 95 m/z (4a) and 107 m/z (5a) display three distinct

262

series within a single GCxGC-TOF-MS run of reduced SRFA. Each series is comprised of

Inset in 2b shows (2c) clusters of prominent

Ions offset by 14 m/z are colored to indicate potential connectivity.

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alicyclic hydrocarbons, with circled regions indicating isomers of the presented molecular

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formula. Insets show possible structures for regions. Mass spectra (3b, 4b, 5b) of compounds

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representative of the series are presented, and insets (3c, 4c, 5c) display prominent fragmentation

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ions. * denotes peak location in chromatograms, molecular ion in spectra, prominent ions in 2c

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It was further determined that the three series in reduced SRFA are comprised of alicyclic

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hydrocarbons.

269

of the spectra from any of the series are consistent with hydrosilylated compounds. Model

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compound work clearly demonstrated that all oxygen containing groups besides phenols are

271

reduced to hydrocarbons. Thus, chemical formulas for these compounds were easily calculated.

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In the series depicted in Figure 3a, the peaks enclosed by a particular region all have the same

273

chemical formula. From these chemical formulae, it was determined that all three of the series

274

are comprised of hydrocarbons with varying degrees of unsaturation (n, where n = number of

275

rings plus double bonds). This assignment is also supported by elution of the three series in the

276

same unsaturated hydrocarbon region established in Figure 1. In model compound work it was

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established that double bonds are only preserved on alicyclic compounds. Therefore, under these

278

conditions, reduction products that are unsaturated hydrocarbons must are bemost likely alicyclic

279

(with or without double bonds).

280

Many of the identified peaks have MS with detectable molecular ions, and none

These alicyclic reduction products are notable for a number of reasons. First, they are

281

broadly distributed over a wide molecular size range.

The most abundant compounds are

282

centered between C13 to C14, but some material appears to be larger than C 20 (Figure 4a).

283

Unfortunately, the analytical method did not preserve molecular ions for compounds >C17. The

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size-range of compounds observed in the reduced sample has important implications for source

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material structure, in that terpenes and terpenoids are the only biomolecules with alicyclic

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backbones of this size.

Terpenoids are biomolecules synthesized from isoprene units that

287

contain five carbons. Thus terpenoid carbon backbones often contain 10, 15, 20, 25 and 30

288

carbons (although, larger terpenoids can be decarboxylated). By convention, terpenes are strictly

289

hydrocarbons, while terpenoids are oxygenated.918

290

Within a given series, alicyclic reduction products are offset by a CH2 group (14 m/z).

291

This methyl offset is not an artifact of the reduction because saturated C-C bonds are stable to

292

the reduction, and must be a feature of native, un-reduced SRFA. In fact, offsets of carboxylic

293

acid groups between individual compounds in Suwannee River Fulvic Acid have been observed

294

using FTICR-MS.7 When reduced, these offsets would appear as a difference of 14 m/z, or the

295

equivalent of a CH2 group in the molecular formula.

296

All compounds within a series are unsaturated to the same degree, and each series varies

297

by one degree of unsaturation, or 2 m/z. All compounds in Figure 3a have one degree of

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unsaturation, meaning that they contain one alicyclic ring. Compounds displayed in Figure 4a

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have two degrees of unsaturation. These compounds may contain a double bond on an alicyclic

300

structure, a fused cyclic structure, or two non-fused rings. Finally, compounds in Figure 5a have

301

three degrees of unsaturation. Possible structures that satisfy each chemical formula and the

302

requirements outlined above are depicted in insets within Figures 3a, 4a and 5a. Unfortunately,

303

exact structures are unavailable because the product MS are too ambiguous. The recovery of

304

compounds offset by 2 m/z (Figures 2b, 3b and 4b) would corroborate other studies which have

305

identified this feature in SRFA.7 However, as established in model compound work, the

306

unsaturation state of products is not necessarily reflective of the unsaturation state of reactants.

307

Thus, while the appearance of series offset by degrees of unsaturation is congruent with other

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previous studies of SRFA, the potential that reduction artifacts contribute to this feature in the

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current dataset must be considered a caveat.

310

There are a large number of peaks within an enclosed region that share the same

311

molecular ion. Mass spectra of these compounds contain the same fragment ions, but the

312

relative abundances of these ions vary. This confirms that each deconvoluted mass spectrum

313

belongs to a unique compound, but that all compounds within a region are structurally related.

314

Therefore, peaks within a region are very likely to be stereoisomers and/or positional isomers.

315

Based on this body of evidence, that compounds in reduced SRFA elute within the

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chromatographic region occupied by unsaturated, alicyclic standard compounds, that double

317

bonds are only stable to theare unlikely to be stable in this reduction system reduction if unless

318

they are present within a ring system, that contiguous (no ether or ester bonds) carbon skeletons

319

between C10 and C17 dominate the chromatogram and are not created during the reduction, it can

320

be concluded that the majority of SRFA carbon that is detectable by GC following this reduction

321

(i.e., 12% of the SRFA carbon) resembles cyclic terpenes. This observation is consistent with

322

previous NMR-based studies that have suggested that terpenoids must be present in SRFA19

323

SRFA9 but these compounds have previously not been chromatographically isolated. To provide

324

some context to the reduction products observed in SRFA, a potential source material was

325

examined.

326

Terpenoids in bald cypress leaves. In the Suwannee River, a major DOM source is organic

327

matter leached from leaf litter.3

328

ran the chemical reduction on freshly fallen leaf litter gathered from Tifton, GA. The leaves are

329

of the genus Ttaxodinium, primarily from the bald-cypress (Ttaxodinium distichum). Bald-

330

cypress is present throughout the entire Suwannee River watershed, and is certain to be a major

19

To confirm the presence of terpenoids in source material we

17



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331

contributor of DOM to SRFA. Although the potential of (leaf colonizing) bacterial and fungal

332

terpenoids must be considered, the intactness of our sample implies that any observed

333

compounds are ultimately plant derived.

334

separated using GCxGC and displayed using the 95 m/z chromatogram (Figure. 6).

The reduced leaf standard was chromatographically

335

The chemical reduction of these source terpenoids to hydrocarbons, and their distribution in

336

the unsaturated region of GCxGC space, confirms that compounds detected in reduced SRFA are

337

indeed derived from terpenes. For example, there are at least two isomers of pinene, a type of

338

bicyclic monoterpene, which are found in the leaves of conifers20 conifers20 and which are

339

present in the extracts of the leaf litter. Under the reaction conditions used, pinene would be

340

expected to reduce to a chemical formula of C10H18. We find that such products are present, and

341

elute near the classification region of reduced SRFA of the same chemical formula. Conifer

342

leaves are also known to contain bicyclic sesquiterpenes such as α-Cadinene2121 (found in leaf

343

extracts). The reduction product of such bicyclic terpenes would have the chemical formula

344

C15H28. Again, we find reduced hydrocarbons of that chemical formula elute in the same

345

GCxGC space as reduced SRFA products. This correspondence does not mean that intact

346

terpenes are present in SRFA. Rather, these smaller terpenoids function as additional model

347

compounds by further establishing the domain of reduced alicyclic compounds in GCxGC space.

348

Such a correspondence is especially reassuring for C15 alicyclic compounds, which are

349

prominent in reduced SRFA, but for which we have no purchased standards.

350

Consistent with previous studies of Taxodium wood,22 Tthe leaf mixture also contained

351

abundant reduction products of the diterpenoid resin acids and triterpenoid phytosterols. It was

352

previously mentioned that the reduction products of 1c are isomers, which elute over a

353

chromatographic region. This region is overlaid in Figure 6. There are at least four (and

18


Page 19 of 28


354

potentially eight or greater) diterpenoid2s020, 2322 that are naturally produced by conifers. These

355

other compounds would also be expected to reduce to isomers, and this is confirmed by the

356

numerous isomers of diterpenoid reduction products that appear in the chromatogram of the

357

reduced leaf mixture (Figure 6).

358 359

360 361

Figure 6. 95 m/z selected- ion chromatogram of reduced bald-cypress leaf litter. Dark markings

362

indicate compound peaks. Regions corresponding to reduced terpenes/terpenoids are colored in

363

orange and labeled with size class. Also overlaid are series from Fig. 4a (black) and selected

364

reduced compounds and hydrocarbon standard from Fig. 1 (red).

365

In contrast to the resin acids, the reduction products of triterpenoid phytosterols appear as

366

discrete, but highly abundant, individual peaks. The small number of peaks indicates that

367

phytosterols are less diverse and/or not (as easily) isomerized by reaction conditions. Reduction

368

products of intact resin acids and phytosterols are not identified in the reduction products of

369

SRFA, which suggests that these compounds are rapidly removed during diagenesis or are

370

transformed into material that escapes detection. Reduced sesterterpenes, which are considered

371

to be rare in nature,243 are also present in the leaf litter sample. The reduced compounds elute in

372

the same GCxGC space as the reduction products of model compound 1e.

19



Page 20 of 28

373

When considering the possible contribution of terpenoids as source material to SRFA, resin

374

acids are an attractive source, especially considering that the distribution of reduced isomers is

375

similar in shape to that of reduced SRFA. However, there are clearly SRFA reduction products

376

larger than C20 (Figure 4a), indicating that phytosterols are also likely to be a significant

377

contributor to SRFA. The cyclic portion of larger terpenoids such as carotenoids could also be a

378

source of the alicyclic compounds observed in SRFA. .

379

material confirms that cyclic terpenoids larger than C15 appear to be a major source for the

380

alicyclic compounds observed in reduced SRFA.

In short, analysis of potential source

381

Finally, in Scheme 1 we present a plausible pathway by which microbial degradation of parent

382

terpenoid compounds could generate the cyclic hydrocarbons observed in reduced SRFA. We

383

used the resin acid Abietic acid (1j) as our precursor model compound. Partially degraded

384

abietanes a and b have been observed as bacterial metabolites of 1j.254 These metabolites only

385

differ by one carbon number, which is the result of decarboxylation at the C-4 position. Further

386

degradation is hypothesized to occur via a c-ring cleavage, catalyzed by a known meta-cleavage

387

dioxygenase,2254 producing 2-isopropyl malic acid (c) in the process. However, no degradation

388

products beyond a and b have been reported on in the literature.

20


Page 21 of 28


389 390

Scheme 1. Hypothetical degradation pathway from cyclic terpenoid to proposed metabolites

391

(components of native SRFA), which then could be further degraded to produce the compounds

392

observed in reduced SRFA.

393

We suggest that further metabolites (d, e) could exist that are either not amenable to recovery,

394

or are extremely short lived. Such bicyclic metabolites could degrade further via a biological

395

Baeyer-Villiger oxidation, forming f and g. The enzyme which catalyzes this reaction is non-

396

specific and capable of oxidizing a large range of cyclic substrates.265 These could then be

397

environmentally hydrolyzed, forming h and i, and/or potentially ester linked into larger

398

compounds.

Reduction of these hypothetical metabolites (d, e, h, i) could produce carbon

21



Page 22 of 28

399

backbones of the size and unsaturation observed in reduced SRFA. Although speculative, it

400

appears that degradation via decarboxylation (and then reduction) is the simplest explanation for

401

the observed CH2 offset of products in reduced SRFA.

402

Molecular Diversity and Future DirectionsMethod contributions to DOM analysis.

403

In a greatly simplified framework, the extreme molecular diversity of NOM can be divided into

404

compositional and structural diversity.We have effectively shown that chemical reduction

405

followed by GCxGC analysis of SRFA allows for moderate interrogation of both structural and

406

compositional diversity, where C compositional diversity refers to the diversity of molecular

407

formulae within a mixture of compounds, and s. Structural diversity refers to the arrangement of

408

elements within a single compound. Both are indicators of source and diagenetic history. We

409

have effectively shown that chemical reduction followed by GCxGC analysis of SRFA allows

410

for moderate interrogation of both structural and compositional diversity.

411

Although capable of revealing compositional diversity in the reaction products, compositional

412

diversity in native SRFA is not directly supported. As the reduction method is comprehensive, it

413

is not possible to determine how many oxygenated functional groups were reduced to form the

414

observed compounds, and therefore it is impossible to extrapolate the exact original composition

415

of reduced compounds. However, we can be certain that direct carbon-carbon bonds were not

416

broken or created during the reduction.

417

compositional diversity, in the context of carbon skeletons, in unreduced SRFA. The distribution

418

of reduced compounds has a maximum in intensity at a molecular formula of C14H26. Unreduced

419

SRFA has a peak maximum at around 350 m/z, which would correspond to a carbon number per

420

molecule at ~15-20.0.2626 It is therefore unlikely that individual SRFA compounds would contain

This allows us to make a reasonable argument for

22


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Page 23 of 28


421

more than one degraded terpenoid backbone. If true, then the observed compositional diversity

422

in reduced SRFA is in part a result of the compositional diversity of unreduced SRFA.

423

There is a great deal of structural diversity present within the UCM, supported by the many

424

isomers identified for each region of a given chemical formula. As a caveat, an unknown

425

amount of diversity could be an artifact of catalyst induced isomerization. However this effect

426

has only been observed in fused cyclic model compounds. The series displayed in Figure 2a are

427

alicyclic, but contain only one ring and no double bonds. Thus, the many compounds observed

428

within each region are certainly not artifacts of the reduction. This demonstrates structural

429

variation within compositionally identical carbon backbones of reduced SRFA, and therefore

430

native SRFA. Broad signals in NMR spectra have been attributed to structural diversity, but to

431

the best of our knowledge this is the first time that structurally distinct isomers have been

432

recovered from SRFA.

433

The isolation of a significant fraction of terpenoid-derived material indicates that all classes of

434

biomolecules are not preserved equally within humic substances. This observation is striking,

435

considering that terpenoids are likely to account for only a small percentage of total organic

436

matter input.

437

preferentially preserved within SRFA.

438

sediments28 is well known, but the current findings support long-term accumulation of these

439

biomarkers in the dissolved phase. Further studies are necessary to confirm if this trend is also

440

observed across different ecological settings, including marine environments.

The results presented here indicate that terpenoid derived compounds are The preferential accumulation of terpenoids in

441 442

ASSOCIATED CONTENT

23



Page 24 of 28

443

Supporting Information. Previously published mechanism of chemical reduction; rationale for

444

GCxGC geography; structure rational for produced isomers. This material is available free of

445

charge via the internet at http://pubs.acs.org.

446

AUTHOR INFORMATION

447

Corresponding Author

448

* email: [email protected].

449

ACKNOWLEDGMENTS

450

The authors thank the National Science Foundation for funding (NSF OCE-1155269 to LIA), as

451

well as the following researchers for inspiration, chemical insights, chromatographic support

452

and foliage: Cameron McIntyre, Vladimir Gevorgyan, Rama Nimmagadda, Brad Allpike, Jack

453

Cochran, Christoph Aeppli, Bob Nelson, Andrew Mehring and Casey Harris.

454 455 456 457 458

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7. Stenson, A. C.; Marshall, A. G.; Cooper, W. T. Exact masses and chemical formulas of

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Direct Identification of Diverse Alicyclic Terpenoids in Suwannee

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