1
Contents
Abstract ... 1
Chapter 1 ... 2
Introduction ... 2Chapter 2 ... 12
Experimental section... 12
2.1 Reagents and materials ... 12
2.2 Medium-polar fraction HOV Pig2 C1... 12
2.3 Equipment and instrumentation ... 13
2.3.1 Sonic Bath ... 13
2.3.2 HPLC-UV ... 13
2.3.3 Spectrofluorophotometer ... 13
2.4 Esterification methods using Fmoc protected amino acids ... 14
2.4.1. Esterification of a model lipid ... 14
2.4.2 Esterification of cholesterol and menthol ... 16
2.4.3 Esterification of diols ... 16
2.4.4 Esterification of composed mixtures of alcohols ... 17
2.4.5 Esterification of HOV Pig2 C1 sample ... 17
2
Chapter 3 ... 19
Results and discussion... 19
3.1. Development of the derivatization protocol using Fmoc protected amino acids and 1-octadecanol as a model lipid compound ... 19
3.1.1 Detection of unreacted 1-octadecanol ... 29
3.1.2 HPLC-UV-MS ... 31
3.2 Esterification reaction between sterols and Fmoc amino acid ... 34
3.3 Esterification reaction between diols and Fmoc amino acids ... 36
3.4 Esterification of composed mixtures of alcohols ... 38
3.5 Esterification of HOV Pig2 C1 sample ... 40
Chapter 4 ... 48
Conclusions ... 48
Appendix ... 50
1
Abstract
In the last decade, a new biomarker paleotemperature approach based on the relative abundances of glycerol dialkyl glycerol tetraethers (GDGTs) has been developed that complements and extends the capabilities of the alkenone proxy. GDGTs are relatively large (up to 86 carbon atoms) membrane lipids produced by Archaea and some Bacteria. They are common, typically abundant, easy-to-analyze lipids found in a variety of environments. Such attributes make GDGTs a promising universal tool for estimating temperatures in the geologic past, especially in deep geologic time.
In this work, a method to increase the detection of GDGTs to allow a better screening of the lipid cores based on the Steglich esterification reaction has been explored.
The following thesis concerns the efficiency of the derivatization approach. The esterification was tested first on a model lipid, then on various molecules with different structure, such as sterols, diols and also a composed mixture. The derivatization reaction had been tested on a medium polar fraction sample taken from a natural loam soil and analysed by liquid chromatography-mass spectrometry.
2
Chapter 1
Introduction
In the last few years, the growth and expansion of a new technique for estimating paleotemperatures based on the use of biomarkers, such as organism-specific found in sedimentary organic matter, were observed. Biomarkers improve the reconstruction of paleoenvironments in geologic history, due to a different set of diagenetic alterations, and they may be well preserved in sedimentary environments. The principle of this biomarker-based paleothermometers is that the microorganisms adjust the rigidity of their cell membrane structures by altering the number of double bonds, rings or branches in response to environmental temperature1,2.
In the past, the biological world was easily divided into prokaryotes and eukaryotes based on the absence or presence, respectively, of a membrane-limited nucleus. Carl Woese and George Fox identified two prokaryotic kingdoms, labeling these Eubacteria and Archaebacteria3. Since Archaebacteria are not bacteria and this term was confusing, it was replaced by the term Archaea. Based on 16S rRNA, Archaea are subdivided into two major kingdoms: Euryarchaeota and Crenarchaeota (Figure 1). The former include methanogens and halophiles, whereas the latter are traditionally referred to the archaea that are thermophilics or hyperthermophilics4.
3 Archaeal membranes are composed of phospho-, glyco- and phospho-glyco-lipids which differ substantially from their bacterial counterparts. Comparing molecular components of archaea with prokaryotes and eukaryotes, extensive chemical differences are observed in the lipids. Unlike the eubacterial and eukaryotic lipids, whose membrane lipid cores consist of fatty acids ester linked to a glycerol moiety, archaeal lipids are mainly isopranyl glycerol ethers6.
The core hydrocarbon chains of archaeal glycerol polar lipids are composed of a phytanyl chain (20 carbons), indicated as glycerol-dialkyl-glycerol-diether (GDGD), or a head-to-head condensation dimer of two phytanyl chains (40 carbons), glycerol-dialkyl-glycerol-tetraether (GDGT)7.
There are two main types of GDGTs: isoprenoidal (isoGDGTs) and the non-isoprenoidal (brGDGTs). The formerly mentioned, just now defined, consist of two head-to-head C40 isoprenoid chains with a varying number of cyclopentane and cyclohexane rings, connected by ether bonds to two terminal glycerol groups (Figure 2). BrGDGTs are structurally similar, but have branched C30 alkyl chains containing 4–6 methyl groups instead of the C40 isoprenoidal chains. Additional polar headgroups, including hexose and phosphate moieties, are attached to the core GDGT structure when the membrane lipid is intact. The main producers of isoGDGTs are members of the Thaumarchaeota.
Indeed, the Thaumarchaeota is the only archaeal phylum known to make the distinctive GDGT crenarchaeol (which has a cyclohexyl ring in addition to the cyclopentyl rings). Thaumarchaeota also produce smaller amounts of a crenarchaeol regioisomer, formally a stereoisomer, (cren’) that plays a crucial role in the temperature predictability of the isoGDGT paleothermometer: relatively more of the regioisomer is observed at higher temperatures. Therefore, a new biomarker paleotemperature approach has been developed. GDGT lipid cores are common, abundant and easy-to-analyze lipids found in difference type of environments (lakes, soils, oceans). These characteristics make GDGTs a promising universal tool for estimating temperatures in the geologic past, especially in deep geologic time.
4 Figure 2 Selection of core structures of the isoGDGTs with mass-to-charge ratios (m/z).
In response to temperature variations, the microbial producers of GDGTs generate compounds with different numbers of rings in their isoprenoid chains: a GDGT with more rings has a higher melting point and is more stable at warm temperatures8. Mesocosm studies
(experiments in which natural seawater is manipulated in the laboratory) show that more of the cyclic isoGDGTs are present in seawater at higher temperatures9,10. An index, represent
the degree of cyclization mathematically, called TEX86 (the TetraEther indeX of 86 carbons)
has been developed for use in palaeoenvironmental assessment11.
The TEX86 index employs relative abundances of specific GDGTs (1, 2, 3 and cren’) to
deduce past sea surface water temperatures (SST) (Eq. 1). TEX86 to SST was empirically
calibrated using a set of 43 modern surface sediments, yielding a strong linear relationship (Eq. 2).
5 TEX86=
([𝐺𝐷𝐺𝑇 − 2] + [𝐺𝐷𝐺𝑇 − 3] + [𝑐𝑟𝑒𝑛′])
([𝐺𝐷𝐺𝑇 − 1] + [𝐺𝐷𝐺𝑇 − 2] + [𝐺𝐷𝐺𝑇 − 3] + [𝑐𝑟𝑒𝑛′]) Eq. 1
TEX86=0.015 × SST + 0.27 (𝑟2 = 0.97, 𝑛 = 43) Eq. 2
Following the development of the linear equation, efforts have been made to transform the original formula in order to constrain the TEX86 proxy and better understand its predictive
ability11,12. Most applications of the relationship between TEX86 and SST utilize the linear
relationships (Figure 3), even though non-linear relationships to SST have been proposed13,14.
Figure 3 Example of cross plot of annual mean World Ocean Database (WOD) at 0 m water depth with TEX86 index values12.
It may be possible that the analytical methods employed in identification and quantification of archaeal GDGTs affect the TEX86 values. The current analytical procedures do not effect
complete resolution of all tetraether structures, leaving essential information inaccessible. The first organic geochemical research into GDGTs made use of chemical degradation techniques. Intact polar and core lipid GDGTs are too large for gas chromatography (GC) and, until relatively recently, the most commonly method15 used was thin layer chromatography (TLC). Fast atom bombardment-mass spectrometry16 and matrix-assisted
6 laser desorption/ionization time-of-flight mass spectrometry17 are less suitable owing to the
complex matrices for sedimentary organic matter.
However, several studies on core lipid GDGT distributions applied high temperature GC18,19
and the coupling of high temperature GC with a time-of-flight mass spectrometer20 were explored.
Nowadays the most widely used technique for analysis of the GDGT lipid cores is high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), which separates compounds by mass and polarity21. The LC approach enables separation of GDGTs with different numbers of cyclopentane moieties (Figure 4). The APCI spectra produce mainly [M+H]+ protonated molecules with minor [M-18+H]+ and [M-74+H]+ ions, the second one is characteristic of loss of the glycerol moiety12,21.
Figure 4 Example of an HPLC–APCI–MS base peak chromatogram of GDGTs22.
0=GDGT-0; 1=GDGT-1; 2=GDGT-2; 3=GDGT-3; 4=Crenarchacol; 4’=Crenarchacol regioisomer.
APCI is an ionization method commonly used in LC-MS, which utilizes gas-phase ion-molecule reactions at atmospheric pressure. APCI can be used for polar and relatively polar compounds with molecular weights up to about 100-1500 Da, producing mono-charged ions that sometimes exhibit some degree of fragmentation. The analyte in solution is introduced into a nebulizer and is converted into an aerosol. The mobile phase and the sample in the gas flow are then vaporized by the heat transferred to the spray droplets in the desolvation
7 chamber and leave the tube as a mixture of the compounds of interest and hot gas. The ionization occurs via a corona discharge electrode (Figure 5).
The ion trap is strictly related to the quadrupole mass filter and it uses the same forces of the quadrupole (electric field and radiofrequency). The ions introduced into the trap are maintained on stable orbits by applying direct current (DC) and radio frequency (RF) potential to the electrodes. To separate ions according their m/z values a RF scan is made. While the RF amplitude increases, ions with higher mass are destabilized and they leave the ion trap moving towards the detector (Figure 5). Moreover, APCI reduces the thermal decomposition of the analyte because of the rapid desolvation and vaporization of the droplets in the initial stages of the ionization.
Figure 5 HPLC-APCI-MS scheme.
Recently the analytical approach has been improved through the use of tandem mass spectrometry (MS/MS or MS2), that involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.
Meaningful information are observed in tandem mass spectrometry, such as the product ion scan and the neutral loss scan. In the product ion scan, a precursor ion is selected in the first stage, allowed to fragment and then all resultant masses are scanned in the second mass analyzer and detected in the detector that is positioned after the second mass analyzer. In a neutral loss scan, the first mass analyzer scans all the masses. The second mass analyzer also scans, but at a set offset from the first mass analyzer. This offset corresponds to a neutral loss that is commonly observed for the class of compounds. In a constant-neutral-loss scan, all precursors that undergo the loss of a specified common neutral are monitored. To obtain
8 this information, both mass analyzers are scanned simultaneously, but with a mass offset that correlates with the mass of the specified neutral. This technique is useful in the selective identification of closely related class of compounds in a mixture.
In this work, a method to increase the detection of GDGTs to allow a better screening of the lipid cores based on the Steglich esterification reaction has been explored (Figure 6).
R OH O
+
R1 OH DCC DMAP cat R O O R1Figure 6 General Steglich esterification reaction scheme.
DCC = dicyclohexylcarbodiimide; DMAP = N,N-Dimethylpyridin-4-amine.
One of the characteristics of the derivatives is the considerably improvement of the signal intensity during LC-APCI-MS, compared with the native lipids. In addition, the polarity of the modified lipids can be controlled.
Several studies employed tert-butyloxycarbonyl (Boc) protected amino acids as the derivatizing agents, while a fluorophore group, opening the possibility of exploiting various detection, has been explored in the following study. Specifically the 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids has been employed (Figure 7).
O
O R
Figure 7 Fmoc (9-fluorenylmethoxycarbonyl) protecting group.
As a first stage, a model lipid was selected for testing the potential of the derivatization approach. Due to low yields during Steglich esterification with literature conditions, ultrasonic bath was then employed. Ultrasonic baths are usually available in organic laboratories. They are the most economical and commercially available sources of ultrasonic irradiation for the chemical laboratory23. The use of ultrasound in chemistry (sonochemistry) offers the synthetic chemist a method of chemical activation, which has broad applications and uses equipment that is relatively inexpensive. The driving force for sonochemistry is
9 cavitation and so a general requirement is that at least one of the phases of the reaction mixture should be a liquid. The normal usage involves the immersion of standard glass reaction vessels into the bath, which provides a fairly even distribution of energy into the reaction medium. The reaction vessel does not need any special adaptation, it can be placed into the bath, and thus an inert atmosphere or pressure can be readily maintained throughout a sonochemical reaction. Temperature control in commercial cleaning baths is generally poor and so the system may require additional thermostatic control.
In order to verify the presence of unreacted reagents at the end of the esterification reaction, a crucial technique has been employed during this work: charged aerosol detection (CAD). CAD allows non-volatile and non-chromophoric analytes to be detected during LC analysis, essential to reveal the presence of unreacted fatty alcohol employed. This technique has several advantages such as the application to both HPLC/UHPLC system and being compatible with gradient conditions, great sensitivities and consistent response independent from chemical structure. The detector measures charge that is associated and in direct proportion to the amount of analyte particles.
The charged aerosol detection process starts with nebulizing the LC column eluent that produces droplets. The largest are removed, while solvent is evaporated from the smaller droplets to produce dried analyte particles.
The analyte particles move to a reaction chamber where they collide with a stream of positively charged gas ions. The positive charges are transferred to the analyte particles. Particle size is in direct proportion to analyte concentration and the larger are the particles, the greater is the charge.
The charged particles then move out of the chamber to a collector while an ion trap remove excess high mobility gas ions. Once in the collector, charge is measured by a sensitive electrometer. This generate a signal that is in direct proportion to the quantity of the analyte (Figure 8).
10 Figure 8 CAD scheme.
The derivatization reaction had been tested on a medium polar fraction sample taken from a natural loam soil and analysed by liquid chromatography-mass spectrometry. The molecular structures of the interested sterols are shown in Figure 9.
Cholesterol O H Brassicasterol O H
11 Campesterol O H Stigmasterol O H β-sitosterol O H Stigmastanol O H Cycloartenol O H
12
Chapter 2
Experimental section 2.1 Reagents and materials
A solution of 9-fluorenylmethoxycarbonyl (Fmoc) protected amino acids and tert-butyloxycarbonyl (Boc) protected amino acids: phenylalanine (Phe), Fmoc-glycine (Fmoc-Gly), Fmoc-proline (Fmoc-Pro), Fmoc-tryptophan (Fmoc-Trp), Fmoc and lysine (Fmoc-Lys(Boc)), phenylalanine (Phe), glycine (Gly), Boc-proline (Boc-Pro) and Boc-tryptophan (Boc-Trp) were purchased from Sigma-Aldrich (UK). 1-hexadecanol, 1-heptadecanol, 1-octadecanol, 1,12-dodecandiol, 1,16-hexadecandiol, 11-bromo-1-undecandiol, 1,2-dihydroxynaphtalene, 1,3-dihydroxynaphtalene,
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1-hydroxybenzotriazole hydrate (HOBt) and N,N-Dimethylpyridin-4-amine (DMAP) were purchased from Sigma-Aldrich (UK). Menthol and quinol were purchased from Fisons (UK). Cholesterol was purchased from BDH Biochemicals (UK).
The following reagents, methanol (MeOH), acetonitrile (ACN), chloroform, hydrochloric acid (HCl), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), 2-propanol (IPA), ethyl
acetate, toluene, 1,2-dichloroethan, diethyl ether (Et2O) and dichloromethane (DCM) were
at least chromatography grade. Doubly distilled water was obtained using Millipore Ultra-Pure water filtration system (Millipore, UK). Silica gel for flash chromatography was of pore size 60 Å and 200-400 mesh particle size (Sigma, UK). All reagents were used as purchased and without any purification.
Thin layer chromatography plates aluminium sheets Silica Gel 60 F254 and Silica gel for column chromatography 60 Å, 230-400 mesh particle size were purchased from Sigma-Aldrich (UK).
2.2 Medium-polar fraction HOV Pig2 C1
A medium polar chromatographic fraction was provided from the InterArChive project (private communication, Scott Hicks, BJK research group member)24. Organic residues had been extracted from the soil sample (Hovingham Experimental piglet burials) and
13 fractionated according to polarity. The C1 is a control taken of the natural loam soil of the site.
2.3 Equipment and instrumentation 2.3.1 Sonic Bath
The sonic bath reactions were performed using a Decon FS100B Heated Ultrasonic Bath without controlling the temperature. The frequency was set on 35-45 kHz.
2.3.2 HPLC-UV
Reverse phase high performance liquid chromatography (RP-HPLC-UV) was performed using two different system. A Dionex (Sunnyvale, CA, USA) Ultimate 3000 RSLC liquid chromatography system was coupled to a DAD detector and the wavelength was set on 262 nm. It was coupled to a Bruker (Coventry, UK) HCTultra ETD II ion trap mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source operated in positive ion mode. The MS scan range was set to m/z 80-1500 and the base peak ion in each scan was selected for collision induced ionization (CID) using an isolation width of m/z 3 and fragmentation amplitude of 5.0 V. The HPLC system was also coupled to a Dionex (Sunnyvale, CA, USA) Corona ultra RS Charged Aerosol Detector (CAD).
An Agilent (Santa Clara, CA, USA) 1100 HPLC system was coupled to a UV detector and the wavelength was set on 262 nm. It was coupled in series with a Jasco (Halifax, NS Canada) FP-920 Intelligent Fluorescence detector and the excitation an emission wavelength were set on 262 and 315 nm.
2.3.3 Spectrofluorophotometer
A Shimadzu RF-5301 was used in spectrofluorophotometer study. The light source was a 150W Xenon lamp. The wavelength scanning was 7-step selection of Survey (about 5500 nm/min), Super (about 3000 nm/min), Very Fast, Fast, Medium, Slow and Very Slow. The wavelength slewing speed was about 20,000 nm/min. The response was 8-step selection of 0.02, 0.03, 0.1, 0.25, 0.5, 2, 4, and 8 seconds for 98% of the full scale. The excitation an emission wavelength were set on 262 and 315 nm whit a ±1.5 nm accuracy.
14 2.4 Esterification methods using Fmoc protected amino acids
2.4.1. Esterification of a model lipid
The fatty alcohol 1-octadecanol was selected as a model lipid for testing the potential of the derivatization approach and six procedures were developed.
Procedure 1
A solution of 1-octadecanol (13.5 mg, 0.05 mmol), Fmoc protected amino acid (0.05 mmol, 1 eq) and DMAP (6.0 mg, 0.05 mmol, 1 eq) in DCM (2 mL) was cooled while stirring with a stir bar in an ice bath for 5 min. EDC (19.2 mg, 0.1 mmol, 2 eq) was added and after 15 min the ice bath was removed and the mixture allowed to warm to ambient temperature overnight (18 h). The reaction mixture was evaporated to dryness in vacuo, the residue was dissolved in ethyl acetate and water (1:1, v/v, 2 × 5 mL) and transferred into a separating funnel. The aqueous phase was discarded and the organic phase washed with HCl (5 %, v/v, 2 × 5 mL), saturated NaHCO3 (2 × 5 mL) and saturated NaCl (2 × 5 mL), before being dried
by passing it through a small column of Na2SO4, the eluent was reduced to dryness under a
gentle stream of nitrogen (N2). The product was isolated on a silica gel column (5 × 50 mm),
eluted with 1.3 column volumes of chloroform : methanol (99:1, v/v) and the solvent was evaporated to yield the pure product.
Procedure 2
A solution of 1-octadecanol (13.5 mg, 0.05 mmol), Fmoc protected amino acid (0.075 mmol, 1.5 eq), DMAP (12.2 mg, 0.1 mmol, 2 eq) and EDC (19.2 mg, 0.1 mmol, 2 eq) in DCM (2 mL) in a conical thick wall glass vial (Reactivial, Thermo) was placed in a sonic bath for 3 h without controlling the temperature. On completion, the reaction mixture was passed through a small silica gel column (5 × 25 mm) to retain the highly polar compounds and evaporated under a gentle stream of nitrogen (N2). The product was isolated on a silica gel
column (5 × 50 mm) and eluted with 2.2 column volumes of 1,2-dichloroethane : methanol (98:2, v/v) and with 1.8 column volumes of 1,2-dichloroethane : methanol (95:5, v/v) for octadecanyl Fmoc-Lys(Boc) ester and the solvent was evaporated to yield the pure product.
15
Procedure 3
The procedure was identical to procedure 2 except of HOBt (13.5 mg, 0.1 mmol, 2 eq) was added to the reaction mixture to investigate its impact on the conversion efficiency.
Procedure 4
The procedure was identical to procedure 2 except that squat form two-dram (7 mL) glass sample vials were used instead of Reactivial’s and the reaction mixture was placed in a sonic bath up to 30 min (except 1 h for Trp) without controlling the temperature.
Procedure 5
The procedure was identical to procedure 4 except of HOBt (13.5 mg, 0.1 mmol, 2 eq) was used instead of DMAP to compare the coupling system.
Procedure 6
A solution of 1-octadecanol (9.3 mg, 0.025 mmol), Fmoc protected amino acid (0.05 mmol, 2 eq), DMAP (15.27 mg, 0.125 mmol, 5 eq) and EDC (19.17 mg, 0.125 mmol, 5 eq) in DCM (1 mL) in an autosampler vial (10 mm clear screw thread autosampler vial, Thermo) was placed in a sonic bath for 2 h without controlling the temperature. The cleaning step was identical to procedure 2.
The Fmoc amino acid octadecanyl esters were dissolved in methanol : chloroform (9:1), sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 100 mm, Dionex, Canada) maintained at 30°C and eluted at a flow rate of 0.7 mL/min with the following gradient: 3 min from 5 to 2% B and isocratic for 1 min (A = acetonitrile and B = 0.5% acetic acid in water).
To verify the presence of unreacted alcohol with the CAD detector, Fmoc amino acid octadecanyl esters were injected onto a Dionex Acclaim column C18 (2.1 × 50 mm, Dionex, Canada) maintained at 25°C and eluted at a flow rate of 0.5 mL/min with the following gradient: isocraticaly at 5% B for 2 min, followed by a gradient to 2% in 2.5 min, then to
16 100% A in 0.5 min and isocratic for 4 min (A = acetonitrile and B = 0.5% acetic acid in water).
2.4.2 Esterification of cholesterol and menthol
The sterols were esterificated following procedure 6, but the reaction needed 4 h to be complete.
The Fmoc amino acid cholesteryl esters were dissolved in methanol : chloroform (9:1), sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 50 mm, Dionex, Canada) maintained at 30°C and eluted at a flow rate of 0.7 mL/min with the following gradient: 5 min from 5 to 2% B and from 10 to 35% C (A = methanol, B = 0.5% acetic acid in water and C = ethyl acetate).
The Fmoc amino acid menthyl esters were dissolved in methanol:chloroform (9:1), sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 50 mm, Dionex, Canada) maintained at 50°C and eluted at a flow rate of 0.5 mL/min with the following gradient: 1 min hold at 40%B, then 5 min gradient from 40 to 5% B (A = methanol, B = 0.5% acetic acid in water).
2.4.3 Esterification of diols
1,11-undecandiol was prepared from 11-bromo-1-undecanol and sodium hydroxide by SN2
nucleophilic substitution.
A solution of alcohol (0.0125 mmol), Fmoc protected amino acid (0.05 mmol, 4 eq), DMAP (15.3 mg, 0.125 mmol, 10 eq) and EDC (23.9 mg, 0.125 mmol, 10 eq) in DCM (2 mL) in squat form two dram (7 mL) glass sample was placed in a sonic bath for 2 h without controlling the temperature. On completion, the reaction mixture was passed through a small silica gel column (5 × 25 mm) to retain the highly polar compounds and evaporated under a gentle stream of nitrogen (N2). The product was isolated on a silica gel column (5 × 50 mm)
and eluted with 3 column volumes of 1,2-dichloroethane : methanol (95:5, v/v) and the solvent was evaporated to yield the pure product.
The Fmoc amino acid esters were dissolved in methanol:chloroform (9:1), sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 50 mm, Dionex, Canada)
17 maintained at 30°C and eluted at a flow rate of 0.7 mL/min with the following gradient: 3.4 min from 25 to 95% B and isocratic for 2 min (A = 10% IPA in MeOH and B = 0.5% acetic acid in water).
2.4.4 Esterification of composed mixtures of alcohols
A mixture of equimolar amounts of 1-hexadecanol, 1-heptadecanol, cholesterol and menthol was esterified with Fmoc-Phe following procedure 6.
A mixture of equimolar amounts of 1-hexadecanol, 1-heptadecanol, cholesterol, menthol, quinol, 1,2-dihydroxynaphtalene and 1,3-dihydroxynaphtalene was esterified with Fmoc-Phe following procedure 6.
The Fmoc amino acid esters were dissolved in methanol:chloroform (9:1), sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 50 mm, Dionex, Canada) maintained at 45°C and eluted at a flow rate of 0.5 mL/min with the following gradient: 1 min hold at 55% B, then a 4 min gradient from 55 to 45% B, isocratic for 1 min and a gradient from 45 to 5% B in 6 min and isocratic for 2 min (A = 25% IPA in acetonitrile and B = water:acetonitrile 1:1).
2.4.5 Esterification of HOV Pig2 C1 sample
The medium polar fraction HOV Pig2 C1 was dissolved in DCM:MeOH 1:1 and was split into three aliquots, each of which was dried under a gentle stream of N2. Esterified employed
three different derivatization agents: Fmoc-Phe, Fmoc-Pro and Fmoc-Lys(B). The solution of the HOV Pig2 C1 medium polar fraction (0.25 nmol, based on an average molar mass of 300 g/mol), Fmoc amino acids (0.625 nmol, 2.5 eq), DMAP (91.6 µg, 0.75 nmol, 3 eq) and EDC (143.8 µg, 0.75 nmol, 3 eq) in DCM (1 mL) were placed in an autosampler vial and placed in a sonic bath for 2 h without controlling the temperature. On completion, the reaction mixture was passed through a small silica gel column (5 × 25 mm) to retain the highly polar compounds, and evaporated under a gentle stream of nitrogen (N2).
The Fmoc amino acid esters were dissolved in methanol:chloroform (9:1), sonicated for 5 min and injected onto a ACE Ultracore 2.5 column Super C18 (3 × 150 mm, Advanced Chromatography Technologies Ltd, UK) maintained at 50°C and eluted at a flow rate of 0.7 mL/min with the following gradient: 1 min hold at 12% B and 15% C, then a gradient from
18 12 to 6% B and from 15 to 35% C in 11.5 min, isocraticaly B and from 35 to 45% C in 4 min, then from 45 to 60% C in 2 min and isocratic for 2 min (A = methanol, B = 0.5% acetic acid in water and C = ethyl acetate).
2.4.6 Methylation of Fmoc protected amino acids
Fmoc protected amino acid (0.025 mmol) was dissolved in 1 ml of toluene:methanol (9:1) and trimethylsilyldiazomethane 2M in hexane (0.1 mmol, 4 eq) was added. The mixture was reacted in a loosely capped vial for 30 min at room temperature. The reaction was performed in a loosely capped vial since nitrogen gas formed during the reaction. On completion, the product was isolated on a silica gel column (5 × 50 mm) eluted with 3 column volumes of 99:1 chloroform:ethyl acetate, then the reaction product was dried under a gentle stream of nitrogen (N2).
The Fmoc amino acid methylated esters were dissolved in methanol, sonicated for 5 min and injected onto a Dionex Acclaim column C18 (2.1 × 50 mm) maintained at 45°C and eluted at a flow rate of 0.4 mL/min isocratic at 35% B for 10 min, (A = methanol and B = 0.5% acetic acid in water).
19
Chapter 3
Results and discussion
3.1. Development of the derivatization protocol using Fmoc protected amino acids and 1-octadecanol as a model lipid compound
The Steglich reaction (Figure 10) is an easy condensation reaction where a carbodiimide is employed to effect coupling of a carboxylic acid and an alcohol to produce an ester25. This reaction was used to modify the hydroxyl functionalities of alcohols to improve their detection. The Steglich reaction does not require expensive catalysts and takes place at room temperature. Differently from what was expected, in the present study, the reaction was not fast and the yield was rather low with respect to N-Boc protected amino acid reactions26.
R O -O
+
N N R1 R2 R O N -R2 O N R1 R O NHR2 O N+ R1 H N R O N+ N+ R O N R O O R' HO-R' -DMAP -H+ N+ N H N N N N : R O O H : -urea H+Figure 10 Steglich esterification reaction.
Fmoc protected amino acids were selected as the derivatizing agent because of their ready availability and the high fluorescence activity of the fluorenyl group, allowing the chance to employ UV detector besides MS.
Fmoc-Lys(Boc) was selected thanks to the combination of two protecting groups: Fmoc and Boc. One characteristic of the N-Boc group is the potential to produce high intensity signals in MS and the possibility of charge leads to possibilities for deprotection.
The amino acids in their protected form reveal relatively apolar character allowing the opportunity for exploration of reversed phase separation techniques.
20 As a model lipid for testing the potential of the derivatization approach, the fatty alcohol, 1-octadecanol (1) was selected, thanks to its ready availability and its low intensity ionization by APCI. In the reactions investigated, the protected amino acid and 1 were coupled using EDC as the carbodiimide condensing agent and DMAP as the catalyst (Figures 11 and 12).
O N O O O R1 R2 OH 16 16
+
O R1 N O O O H R2 EDC, DMAP DCM 1 2 R1=Ph R2=H 3 R1=H R2=H 4 R1R2= (CH2)3 5 R1= C H3 N H R2=H 6 R1=Ph R2=H 7 R1=H R2=H 8 R1R2= (CH2)3 9 R1= C H3 N H R2=HFigure 11 General scheme of the Steglich esterification reaction between the model lipid (1) and selected Fmoc
protected amino acids using EDC/DMAP coupling system. Fmoc protected amino acids used were: phenylalanine (2), glycine (3), proline (4) and tryptophan (5).
O O O O O NH NH O 16 OH 16
+
O O O O O NH NH OH EDC, DMAP DCM 1 10 11Figure 12 General scheme of the Steglich esterification reaction between the model lipid (1) and
21 Six procedures were explored in order to optimize the percentage of yield of the ester product.
In procedure 1, the Steglich reaction, the mechanism is well known25,27. The reaction
proceeds through an intermediate O-isoacyl urea, formed by nucleophilic addition to the carbodiimide. On attack of the alcohol, the O-isoacyl urea is transformed into the final ester product and a urea by-product.
The ester products, separated from unreacted reagents by a liquid-liquid extraction described in the experimental section, were purified on silica gel and the yields to provide the results shown in Table 1. The presence of unreacted reagents, confirmed by thin layer chromatography (TLC, aluminium sheets Silica gel 60 F254) developed using DCM:MeOH:AcOH (95:5:1), indicate that the reaction conditions were not optimal, thus resulting in a low yield of ester products.
Table 1 Yields of ester products formed in Steglich esterification reaction (Procedure 1) of 1-octadecanol (1)
with selected Fmoc protected amino acids (2-5) using EDC/DMAP in DCM.
Entry Ester product Yield (%)
1 (6) Octadecanyl Fmoc-Phe 79 2 (7) Octadecanyl Fmoc-Gly 64 3 (8) Octadecanyl Fmoc-Pro 65 4 (9) Octadecanyl Fmoc-Trp 67
The reasons why the yield is low may be the large dimensions of the 9-fluorenylmethoxycarbonyl group and the initial cooling to 0°C for 20 min probably decreased the kinetic energy of the reactants, leading to the reaction time at room temperature not being sufficient. Because of the slow warming of the reaction mixture, allowing longer time for the reaction to warm does not resolve the issue, indeed there were no changes in terms of yield over 18 h. Another possible reason for the low yield may be the aqueous work-up step. Therefore another protocol for isolating the product was tested, which consisted in passing the crude solution through a short column of silica gel, as discussed in the experimental section. When the procedure was tested on a replicate of entry 4, EDC, DMAP and unreacted 5 were retained by the column and the yield of ester product 9 was increased to 79%.
22 To determine the effective yield, the preparation of 7 was selected for this study. The final mixture was split in three portions: one not treated, one purified by passing it through a short silica gel column and the last one methylated using trimethylsilyldiazomethane. The latter operation was effectuated to methylate the amino acid and after injection in HPLC-UV. The concentration of the unreacted 1 can be determinated, knowing the effective conversion degree of the reaction.
Five standard solutions of differing concentrations of Fmoc-Gly-methyl ester were prepared (1, 5, 10, 50 and 100 µg/mL), injected on HPLC-UV in triplicate using the program for methylated Fmoc amino acid esters separation (Table 2) and the calibration curve was obtained (Figure 13).
Figure 13 Calibration curve of Fmoc-Gly methyl ester (n=3), the equation and the correlation coefficient.
Similarly, the calibration curve of 7 (Figure 14) was determinated injecting in triplicates five different concentration solutions (20, 40, 100, 200 and 400 µg/mL) of the isolated ester.
Figure 14 Calibration curve of octadecanyl ester Fmoc-Gly 7 (n=3), the equation curve and the correlation
coefficient. y = 15,092x - 1,2689 R² = 0,9999 0,0 500,0 1000,0 1500,0 0 20 40 60 80 100 120 A re a mA u *s Concentration µg/mL y = 23,155x + 113,39 R² = 0,9989 0,0 2000,0 4000,0 6000,0 8000,0 10000,0 0 100 200 300 400 500 A re a mA u *s Concentration µg/mL
23 After dilution, the crude and the purified sample were injected using the program developed for octadecanyl esters separation, while the methylated sample was first injected using the same program and then analized with the methyl ester method (Table 2).
Table 2 Elution program of octadecanyl esters and methyl esters in HPLC
Separation program name T (°C) Flow rate (ml/min)
% B (0.5% AcOH in H2O)
A
Octadecanyl esters 30 0.7 from 5 to 2 % (3 min)
2% isocratic ACN Methyl esters 45 0.4 35 % isocratic (10 min) MeOH
Due to variable results, no replicates of the measurements were carried out. In turn, this hampered the quantification of unreacted 3 by this approach. Therefore, it was evident that an alternative method to verify the presence of unreacted reagent had to be investigated. Thanks to its characteristics, discussed in the introduction, charged aerosol detector (CAD) was employed for further studies.
To increase the yield of product a sonic bath was used with the rest of esterification procedures. Shorter reaction time and increasing the chemical activation are the main advantages in employing sonication.
The derivative Fmoc-Gly (3) was first selected for testing procedure 2. After 2 h of reaction, the reaction mixture was spotted on a TLC plate every 30 min until 3 h and was developed using chloroform : methanol (99:1) to verify if 1 was still present (Figure 15). After 2 h 1 was totally reacted and 7 and another product, possibly an intermediate, were present. After 2.5 h there was no intermediate product change remaining by TLC, hence the reaction is complete within less than 3 h28.
Figure 15 2, 2.5 and 3 h TLC plate of Fmoc-Gly-octadecanyl ester (7) and 1-octadecanol (1) developed using
chloroform : methanol (99:1) Intermediate Fmoc-Gly-octadecanyl ester 1-octadecanol 7 7 7 1 2h 2.5h 3h
24 The procedure was then applied to all of the Fmoc amino acids. After 3 h the ester products were isolated on silica gel to give the yields reported in Table 3. The increase in the yields could be explained by the differences in the work-up process, the use of sonication and the elimination of the cooling step. Using a short silica gel column, instead of the aqueous work-up, reduced the possibility of loss of some product over several isolation steps. Moreover, in the sonic bath the kinetics of the reaction has been probably increased and, thanks to the use of DMAP, the formation of urea intermediate26 has been prevented.
Table 3 Yields of ester products formed in the esterification (procedure 2) of 1 with selected Fmoc protected
amino acids (2-5, 10) using EDC/DMAP in DCM.
Ester product Yield (%)
(6) Octadecanyl Fmoc-Phe 88* (7) Octadecanyl Fmoc-Gly 93
(8) Octadecanyl Fmoc-Pro 95 (9) Octadecanyl Fmoc-Trp 98 (11) Octadecanyl Fmoc-Lys(Boc) 96
*the aqueous work-up was applied
Procedure 3 differs from procedure 2 by the additional use of 1-hydroxybenzotriazole hydrate (HOBt) to investigate its impact on conversion rate. The purpose of HOBt is to minimize the formation of urea29 and prevent degradation and racemization.
Fmoc amino acids 4, 5 and 10 were employed with this procedure. On completion of the reactions, TLC plates were spotted and developed using 1,2-dichloroethane : methanol (98:2) for products 8 and 9, and 1,2-dichloroethane : methanol (95:5) for product 11. TLC plates showed the presence of the ester products, unreacted 1 and, lastly, another product, possibly the ester formed by reaction of the Fmoc amino acid and HOBt28,30 (Figure 16).
The latter would result from the incomplete replacement of HOBt by 1, in the intermediate activated ester 12 which forms under these conditions (Figure 17).
25 Figure 16 TLC plate of the ester Fmoc-Pro octadecanyl (8), Fmoc-Trp octadecanyl (9), Fmoc-Lys(Boc)
octadecanyl (11) and 1-octadecanol (1) developed using (a) 1,2-dichloroethane:methanol (98:2) and (b)1,2-dichloroethane:methanol (95:5). R OH O
+
N N R2 R1 R O N -R2 O N R1 N N N OH N N N O R O 12Figure 17 Formation of active ester 12 with HOBt/EDC28.
The ester products were purified on silica gel to give the yields reported in Table 4. The yield was slightly decreased with respect to procedure 2, probably as a consequence of the competing action of HOBt.
Table 4 Yields of ester products formed in the esterification (procedure 3) of 1 with selected Fmoc protected
amino acids (8, 9 and 10) using EDC/DMAP and HOBt in DCM.
Ester product Yield (%)
(8) Octadecanyl Fmoc-Pro 91 (9) Octadecanyl Fmoc-Trp 93 (11) Octadecanyl Fmoc-Lys(Boc) 91
Procedure 4 was performed using squat form glass sample vial (7 mL) instead of the thick conical vial, to examine if the reaction could be completed in less time. The use of the squat form glass sample vial instead of the Reactivial, should increase the kinetics of the reaction,
Fmoc-aa octadecanyl ester 1-octadecanol HOBt ester Fmoc-aa-octadecanyl ester 1-octadecanol HOBt ester 8 9 1 (a) 11 1 (b)
26 due to a better propagation of sonic waves. Esterification reaction was indeed faster than the previous one. Fmoc amino acids and Boc amino acids were employed during this procedure and each reaction was repeated three times.
The esterification went to completion in 30 min for all the amino acids except Fmoc- and Boc-Trp octadecanyl esters, that required 1 h. The ester products were purified on silica gel to give the yields reported in Table 5.
Table 5 Yields of ester products formed in esterification (procedure 4) of 1 and with N-Fmoc and N-Boc
protected amino acids using EDC/DMAP in DCM.
Ester product Yield (%)
1 2 3 (6) Octadecanyl Fmoc-Phe 94 93 92 (7) Octadecanyl Fmoc-Gly 96 96 95 (8) Octadecanyl Fmoc-Pro 95 92 94 (9) Octadecanyl Fmoc-Trp 90 97 97 (11) Octadecanyl Fmoc-Lys(Boc) 97 92 96 (13) Octadecanyl Boc-Phe 98 98 98 (14) Octadecanyl Boc-Gly 96 95 95 (15) Octadecanyl Boc-Pro 87 86 86 (16) Octadecanyl Boc-Trp 93 92 93
In procedure 5 HOBt was used as the catalyst in the esterification reaction, under conditions otherwise identical to those of procedure 4 with 2 and 10 as the amino acid components. After 30 min and 1 h, TLC analysed showed the presence of the unreacted alcohol. Therefore, the HOBt system does not work as well as the DMAP one. For this reason, the method was not examined in further datail and the ester yields were not determinated. In order to evaluate if increasing the quantity of the EDC/DMAP coupling agent would increase the yield, procedure 6 employed autosampler vials, with a five-fold excess of the coupling agent instead of two-fold excess used in procedure 4. Two Fmoc amino acids, 2 and 10, were selected to testing the impact on the yield of 6 and 11, respectively. After 2 h in the sonic bath, the TLC plate showed the absence of unreacted 1 for both reactions. The cleaning step was identical to procedure 2 and after ester purification on a short silica gel
27 column the yields were 96% for 6 and 95% for 11. Comparison with the results obtained in procedure 4 indicates that increasing the amount of the coupling agent did not significantly affect the yield of ester product. In this conditions, it is evident that the use of ultrasonic bath and the silica gel column for product formation and isolation respectively are efficient for the purpose. The use of squat form glass sample vials and autosampler vials reduced the reaction time, without impacting negatively on product yield. Moreover, by employing smaller vials, as in procedure 6, less reagents and solvents were used, reducing also purification time.
Even though no substantial difference were observed between procedure 4 and 6, the latter was chosen for further studies owing to the lack of real sample. Therefore, procedure 6 had allowed to reproduce the conditions employed during lipid extract analysis.
The results of the procedures for the formation of the octadecanyl esters of Fmoc and N-Boc protected amino acids, except Procedure 5, are compared in Table 6.
28 Table 6 Summary table of the 6 procedures’ yield (yield in procedure 5 not calculated)
Ester Product
Yield (%) Procedure 1 Procedure 2 Procedure 3 Procedure 4 1 2 3 mean Procedure 6(6) Octadecanyl Fmoc-Phe 79 88 * n/a 94 93 92 93 96
(7) Octadecanyl Fmoc-Gly 64 93 n/a 96 96 95 96 n/a
(8) Octadecanyl Fmoc-Pro 65 95 91 95 92 94 94 n/a
(9) Octadecanyl Fmoc-Trp 67 98 93 90 97 97 95 n/a
(11) Octadecanyl Fmoc-Lys(Boc) n/a 96 91 97 92 96 95 95
(13) Octadecanyl Boc-Phe 94 n/a n/a 98 98 98 98 n/a
(14) Octadecanyl Boc-Gly 112 n/a n/a 96 95 95 95 n/a
(15) Octadecanyl Boc-Pro n/a n/a n/a 87 86 86 86 n/a
(16) Octadecanyl Boc-Trp 88 n/a n/a 93 92 93 93 n/a
* the aqueous work-up was applied instead of the short silica gel column
data from C. Poplawski’s work
n/a – no reaction was carried out under such conditions
29 3.1.1 Detection of unreacted 1-octadecanol
To verify the absence of 1 in the products, HPLC with CAD was used. One of the characteristics of this detector is that it allows non-volatile and non-chromophoric analytes to be detected. Then, CAD was crucial in the following study.
From a stock standard solution of 1, five diluted solutions (1.1, 5.5, 11, 27.5 and 50 µg/mL) were prepared and injected in triplicate on HPLC-UV-CAD to provide the calibration curve shown in Figure 18.
Figure 18 Calibration curve of 1-octadecanol (n=3), with the equation and the correlation coefficient.
Reactions to form 6, 7, 8, 9, 11 were repeated using procedure 4 and 100 µL of crude product solution were taken before passing through silica gel column, dried and reconstituted in 10% chloroform in MeOH, diluted and injected. The chromatograms (Figure 19) shown that unreacted 1 was not present in any of the solutions. The yields obtained after isolation are reported in Table 7. It can be assumed that the yields based on the weight previously calculated are the real yields. Moreover, those results were congruent with the values already obtained.
Table 7 Yields of ester products in repeated procedure 4.
Ester product Yield (%)
(6) Octadecanyl Fmoc-Phe 94 (7) Octadecanyl Fmoc-Gly 96 (8) Octadecanyl Fmoc-Pro 95 (9) Octadecanyl Fmoc-Trp 97 (11) Octadecanyl Fmoc-Lys(Boc) 97 y = 0,0471x - 0,0665 R² = 0,9994 0,00 0,50 1,00 1,50 2,00 2,50 3,00 0 10 20 30 40 50 60 Are a (p A* m in ) Concentration µg/mL
30 Figure 19 Comparison of HPLC-CAD chromatograms octadecanol 1 (a) and crude reaction products (b-7, c-9, d-11, e-6, f-8). (a) (b) (c) (d) (e) (f)
31 The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the signal-to-noise (S/N) ratio method31 and the results are reported in Table 8. A S/N ratio of
three is calculated as LOD and S/N of ten as LOQ. Standard deviation is calculated on six replicates of the lowest concentration.
Table 8 Limit of Detection and Limit of Quantification for 1 by HPLC-CAD
LOD (µg/mL) 0.213 ± 0.003 LOQ (µg/mL) 0.710 ± 0.003
From the results obtained, considering an initial concentration of 1 of 10 µg/mL, the unreacted residual will be 2%, equivalent to a 98% conversion degree.
3.1.2 HPLC-UV-MS
The Fmoc derivatives are suitable both for UV and fluorescence detection. The wavelength maxima are for absorption around 262 nm and for fluorescence emission at 315 nm32. Equimolar solutions of the 5 isolated products were prepared in 10% chloroform in methanol and analysed by HPLC-UV-MS and HPLC-UV-FLD following the HPLC programs described in experimental section.
The elution order of the octadecanyl esters was Gly (7), Trp (9), Fmoc-Lys(Boc) (11), Fmoc-Phe (6) and Fmoc-Pro (8), confirmed also by the MS detection. In particular, one component, octadecanyl Fmoc-Trp (9), exhibited very low fluorescence yield, this being attributed to quenching by its indole moiety33 (Figure 20).
32 Figure 20 Chromatogram with UV (a) and fluorescent (b) detection of octadecanyl Fmoc-amino acids esters.
The octadecanyl esters produced very clean mass spectra, each showing only two ions: the protonated molecule and a fragment ion formed by the loss of the Fmoc group, equivalent to 222 Da. The loss of Fmoc proceeds via Mclafferty type rearrangement34 resulting in a protonated amino acid ester fragment exhibiting relatively high signal intensity in the MS (Figure 21, Table 9). Contrary to the mono Fmoc derivative esters, 11 immediately lost the Boc group (100 Da) in APCI via the mechanism35 shown in Figure 22, as well as loss of Fmoc amide, equivalent to 239 Da (Figure 21).
Table 9 Protonated molecule and fragment ion formed by the loss of the Fmoc group of the octadecanyl esters.
Ester product [M+H]+ [M+H-Fmoc]+
(6) Octadecanyl Fmoc-Phe 640 418 (7) Octadecanyl Fmoc-Gly 550 328 (8) Octadecanyl Fmoc-Pro 590 368 (9) Octadecanyl Fmoc-Trp 679 457 Minutes 0 1 2 3 4 5 6 7 8 9 10 m Volt s 0 10 20 30 40 50 60 70 80 m Volt s 0 10 20 30 40 50 60 70 80 FID 6000 7 9 11 6 8 7 9 11 6 8 (a) (b)
33 Figure 21 Mass spectrum of 6 (a) and 11 (b) with loss of Fmoc and Boc groups.
O O N H H R1 R2 C H3 CH3 CH2
+
H O NH O R2 R2 CO2 R2 N H2 R2+
m=56 Da m=44 DaFigure 22 Loss of Boc mechanism34.
The mass spectrum also shown a loss of 44 Da and 196 Da, which correspond to the fragmentation of Fmoc amide in isocyanic and 9-fluorenemethanol respectively.
The best derivatizing agent had to be selected by taking into account of the conversion rate, UV, fluorescence and MS signal.
A spectrofluorophotometer study on the esters was made. Equimolar solutions of isolated products 6, 7, 8, 9 and 11 were prepared in 10% chloroform in methanol, analysed and compared (Figure 23).
(a)
34 The results revealed that Fmoc-Trp octadecanyl ester gave the lowest intensity signal due to the anticipated quenching by the indole moiety. Fmoc-Gly octadecanyl ester gave the best intensity signal, but considering the low response in MS, this derivatizing agent was rejected in favour of Fmoc-Lys(Boc) octadecanyl ester, 11. Indeed, 10, was the amino acid with the highest intensity during spectrofluorometry and MS.
For further studies, 2 and 10 were mainly tested.
Figure 23 Overlapping of absorption spectra of the 5 isolated octadecanyl esters. λex=262 nm; λex=315 nm.
3.2 Esterification reaction between sterols and Fmoc amino acid
To verify the applicability of the esterification with different structures, including secondary alcohols in bulky molecules, cholesterol and menthol were reacted with 2 and 10 using Procedure 6. Even though there were not any appreciable difference in yield compared with procedure 4, this procedure was applied to reproduce the sample conditions.
The yields were surprisingly low for cholesteryl esters (72% Phe, 16, and 70% Fmoc-Lys(Boc), 17). This is may be explained by the bulky structure of cholesterol compared with the model straight chain alcohol. Unusually, the cholesteryl ester with Fmoc-Lys(Boc) did not behave in MS like the modified model alcohol (Figure 24). It immediately lost the Boc group, but it did not lose 239 Da, equivalent to the Fmoc amide. On the other hand, the loss of Fmoc group is observed in both ester products.
The menthyl ester yields were 86% for Fmoc-Phe (18) and 88% for Fmoc-Lys(Boc) (19). This can be explained because menthol is a very small molecule compared with cholesterol.
35 As with the cholesteryl ester, the mass spectrum of 19 shows the molecular ion with the loss of Boc group (m/z= 507).
Figure 24 Mass spectrum of Fmoc-Lys(Boc) cholesteryl ester (17) without the loss of 239 Da.
In order to evaluate if differences in the response may be due to differences in the absorption coefficient of different composition of solvent gradient, esters products 11, 17 and 19 were employed during a spectrofluorophotometer study (Figure 25).
According to the HPLC gradient program and the polarity of the involved compounds, equimolar solutions of each ester were prepared in three different solvents to investigate the solvation process, hence, to verify if the signal in MS might be affected. For this reason, the solvents selected were: 10% 2-isopropanol in methanol; 10% ethyl acetate in methanol and 10% 2-isopropanol in acetonitrile.
In Figure 25a and 25b, the esters’ absorbance responses were almost the same, therefore the solvation process did not affect the signal intensity. On the contrary, as shown in Figure 25c, the combination of 2-isopropanol and acetonitrile could differentiate and actually amplify the response of the three modified alcohols.
However, employing these solvent compositions, the derivatized alcohols’ signal was not decreased.
36 Figure 25 Absorption spectra of sterols (11, 17 and 19) using (a) 10% IPA in MeOH, (b) 10% EtOAc in MeOH
and (c) 10% IPA in ACN. λex=262 nm; λex=315 nm.
3.3 Esterification reaction between diols and Fmoc amino acids
Diols were selected to verify the applicability of derivatization on molecules with two hydroxyl functionalities. The first diol derivatized with 2 was 1,11-undecandiol, previously prepared from 11-bromo-1-undecanol and sodium hydroxide by SN2 nucleophilic
substitution, purified and isolated. The reaction took 2 h and the TLC showed that two end products were present, confirmed also by chromatography and MS: the monoester and the diester (Figure 26). (a) (b) (c) Octadecanyl Menthyl Cholesteryl
37 Figure 26 Mass chromatogram of modified 1,11-undecandiester Fmoc-Phe and fragmentation of modified
mono- and diester.
Probably the derivatization process was incomplete and the reaction needed more time to reach total conversion.
The Fmoc-Phe modified diester mass spectrum (Figure 27a) showed the loss of 222 Da and 444 Da that correspond to the loss of one and two Fmoc groups respectively. During MS analysis, the modified monoester (Figure 27b) lost one Fmoc group (-222 Da).
Figure 27 Mass spectra of (a) derivatized 1,11-undecandiester (m/z=928) and (b) 1-undecanester (m/z=558).
(a)
38 The pure purchased products 1,12-dodecandiol and 1,16-hexadecandiol were esterificated with 2 and 10. After 2 h the TLC spotted with Fmoc compound showed only one product whereas after chromatography and MS analysis, two final products were observed: the monoester (in small quantity) and the diester. By contrast, 10 reacted to completion, giving the diester as the final product. It may be possible that the reaction with 2 needed more than 2 h to go to completion or the amount of coupling reagents needed to be increase in order to obtain the diester without traces of the monoester.
During MS analysis the Fmoc-Lys(Boc) diesters immediately lost the Boc groups (-100 Da and -200 Da) and then an Fmoc group (-322 Da) as shown in Figure 28. The loss of 239 Da, corresponding to the Fmoc group and ammonia, was not detected. Fmoc-Phe modified diesters behaved in the same way as 1,11-undecandiester, previously discussed, losing first one and then the second Fmoc group (-222 Da and -444 Da).
Figure 28 Mass chromatogram and mass spectrum of Fmoc-Lys(Boc) 1,16-hexadecanyl diester.
3.4 Esterification of composed mixtures of alcohols
In order to test the derivatization efficiency in a composed mixture with more than one analyte, two mixture were prepared: one with and one without aromatic compounds. The two mixtures’ composition has been described in detail in the experimental section. The structure of aromatic compounds are shown in Figure 29.
39 quinol OH OH 1,2-dihydroxynaphthalene OH OH 1,3-dihydroxynaphthalene OH OH
Figure 29 Structure of aromatic compounds in the mixture.
Procedure 6 was applied in this study and 2 was the derivatizing agent tested.
The mixtures were injected in HPLC-MS system and compared. As shown in Figure 30, the elution of the esters in the mixture without aromatic compounds was menthyl first, 16-hexadecanyl, 17-heptadecanyl and cholesteryl as last product. In the case of the aromatic compoundsquinyl eluted as the first compound, then 1,2-naphtalendiyl diester and 1,3-naphtalendiyl diester. While modified cholesterol eluted after the heptadecanyl ester, its derivatives should eluted after medium-long chain fatty alcohols.
The chromatograms show a notable drift, due to the gradient eluent composition. Even though the mixture had an equimolar compositions mixture, cholesterol seems to be in a lower concentration compared to the other compounds present.
40 Figure 30 Chromatogram of mixtures of alcohols. Quinyl (1), 1,2-naphtalendiyl diester (2), menthyl (3),
1,3-naphtalendiyl diester (4), hexadecanyl (5), heptadecanyl (6) and cholesteryl (7). T = 45°C; flow rate = 0.5 mL/min; 1 min isocratic at 55% B, 4 min from 55 to 45% B, 1 min isocratic, 6 min from 45 to 5% B, 2 min isocratic (A = 25% IPA in acetonitrile and B = water:acetonitrile 1:1).
3.5 Esterification of HOV Pig2 C1 sample
The derivatization method was applied to the total lipid extract from a natural loam soil taken from InterArChive experimental burials and previously characterized by gas chromatography mass spectrometry (GC-MS)24. The chromatogram (Figure 31) shows the presence of fatty alcohols (marked as ) and some sterols (marked as *).
The sample, split in three aliquots, was derivatized with three different Fmoc protected amino acids selected for their best characteristics investigated previously: Fmoc-Phe, because of its high conversion rate and good signal in UV, Fmoc-Lys(Boc), because of its very high and clean signal in MS and Fmoc-Pro because of its waxy property and good intensity in MS.
The n-alkanols from C20 to C31 and 4 sterols were identified to be present based on previous GC-MS analysis work24 (Table 10). GC-MS analysis employed a situ derivatization with trimethylsilyldiazomethane.
Mixture without aromatic compounds Mixture with aromatic compounds
1
2 3 4
5 6
41 Table 10 Compounds present in the gas chromatogram (See Figure 31).
Peak Name MW 1 1-eicosanol 298.6 C20 2 1-docosanol 326.6 C22 3 1-tricosanol 340.6 C23 4 1-tetracosanol 354.7 C24 5 1-pentacosanol 368.7 C25 6 1-hexacosanol 382.7 C26 7 1-octacosanol 410.8 C28 8 1-triacontanol 438.8 C30 1* Cholesterol 386.7 Δ5-C27 2* Stigmasterol 412.7 Δ5,22-C29 3* β-sitosterol 414.7 Δ5-C29 4* Stigmastanol 416.7 C29
42 Figure 31 Gas chromatogram24 of HOV Pig2 C1.
42 1 2 3 4 5 7 6 1* 2* 3* 8 4* - fatty alcohol * - sterol
43 The lipid extract derivatized with 10 was selected and analysed by HPLC-MS owing to the greatest signal intensity of Fmoc-Lys(Boc) esters during MS analysis. A very clean mass chromatogram was obtained by refining the data so to exhibit a constant loss of 239 Da, followed by smoothing of the data (Figure 32). By comparison with the components identified in GC-MS, a greater number of sterols and a wider range of alcohols were identified (Table 11).
Table 11 Compounds present in the HPLC-MS chromatogram (See Figure 32).
Peak Name MW 1 1-octadecanol 270.5 C18 2 1-docosanol 326.6 C22 3 1-tricosanol 340.6 C23 4 1-tetracosanol 354.7 C24 5 1-pentacosanol 368.7 C25 6 1-hexacosanol 382.7 C26 7 1-heptacosanol 396.7 C27 8 1-octacosanol 410.8 C28 9 1-nonacosanol 424.8 C29 10 1-triacontanol 438.8 C30 11 1-hentriacontanol 452.8 C31 12 1-dotriacontanol 466.9 C32 13 1-tetratiacontanol 494.9 C34 1* Unknown sterol 372 2* Brassicasterol 398.7 Δ5,22-C27 3* Campesterol 400.7 Δ5-C28 4* Stigmasterol 412.7 Δ5,22-C29 5* β-sitosterol 414.7 Δ5-C29 6* Cycloartenol 426.7 Δ24-C30 7* Unknown sterol 456
44 Figure 32 Mass chromatogram of lipid extract obtained with loss of 239 Da and smoothing. T = 50°C; flow rate = 0.7 mL/min; 1 min isocratic at 12% B and
15% C, 12 min from 12 to 6% B and from 15 to 35% C, 4 min isocratic B and from 35 to 45% C, 2 min from 45 to 60% C, 2 min isocratic (A = methanol, B = 0.5% acetic acid in water and C = ethyl acetate).
44 1* 1 2* 3* 5* 6* 2 3 7* 4 5 6 7 8 9 10 11 12 13 - fatty alcohol * - sterol 4*
45 The mass chromatogram obtained could be split in two parts: the initial one (between 2 and 10 min) containing mainly sterols and the final one (between 10 and 20 min) that comprises mainly linear alcohols.
Initially, C20 n-alkanol, cholesterol and stigmastanol were not identified in the mass chromatogram. These sterols and alcohol coeluted with other components and it was necessary change the gradient during the elution program. Cholesterol (7*) and eicosanol (14) were then identified (Figure 33). This compounds coeluted with campesterol and cycloartenol, respectively.
Figure 33 Detail of a mass chromatogram. Detection of cholesterol (8*) and 1-eicosanol (14) with modified gradient program.
Using stronger solvent, C36 n-alkanol was detected (Figure 34) at the end of the chromatogram, but the separation in the first part of the spectrum was lost.
Figure 34 Mass spectrum of C36 Fmoc-Lys(Boc) ester (m/z=973).
Two unknown sterols were detected. Basing on the molecular weight, 1* might be a compound similar to cholesterol with two unsaturations and lacking a methyl group. 7* might be a related to cycloartenol, e. g. the sterol with 2 more methyl groups or a diol.
8*
3* 14
46 Sterols were in low concentration compared with the linear alcohols. Stigmastanol, identified by GC-MS, was not detected. It may be possible that changing the mobile phase composition could resolve coelutions in the first part of the chromatogram. Alternatively, the absence of this compound may reflect the esterification needing more than 2 h for completion.
In Table 12, a summary of all the compounds identified in GC-MS and HPLC-MS is showed. Table 12 Summary table
Name GC-MS HPLC-MS 1-octadecanol C18 X 1-eicosanol C20 X X 1-docosanol C22 X X 1-tricosanol C23 X X 1-tetracosanol C24 X X 1-pentacosanol C25 X X 1-hexacosanol C26 X 1-heptacosanol C27 X 1-octacosanol C28 X X 1-nonacosanol C29 X 1-triacontanol C30 X X 1-hentriacontanol C31 X 1-dotriacontanol C32 X 1-tetratiacontanol C34 X 1-hexatriacontanol C36 X Cholesterol Δ5-C27 X X Stigmasterol Δ5,22-C29 X X β-sitosterol Δ5-C29 X X Stigmastanol C29 X Brassicasterol Δ5,22-C27 X Campesterol Δ5-C28 X Cycloartenol Δ24-C30 X Unknown sterol (MW=371) X Unknown sterol (MW=456) X
47 Employing the Steglich reaction on the lipid extract allowed the identification of more n-alkanol and various sterols previously not recognised. The detection is improved and the chromatogram obtained was much clearer owing to the removal of many non-alcohol components.
48
Chapter 4
Conclusions
This work has shown that lipid extracts can be modified with a range of N-Fmoc protected amino acids in an easy way by applying the Steglich esterification reaction.
The reaction is improved due to the use of sonic bath and squat form dram glass sample vial. In particular, ultrasonic bath performance does not require special device during the procedure, since the bath employed was not equipped with temperature control. The esterification took place at room temperature and the initial cooling step was removed. The time is then reduced to 4 h at most, instead of 18 h in previous procedures.
Moreover, the isolation of esters is achieved by simple passage through a short silica gel column instead of the aqueous work-up, reducing time, quantity of solvents used and, especially, loss of product during several separation steps.
This developed novel protocol is also suitable for N-Boc protected amino acids. The yields obtained are, in most cases, more than 95%. The conversion of the alcohol is, indeed, nearly quantitative.
The UV/fluorescence detection allows selective targeting via the fluorenyl group of the N-Fmoc protected amino acids.
The study on the model lipid allowed understanding of the fragmentation mechanisms during the ionization in the APCI source for the various Fmoc amino acids tested.
MS and fluorescence intensity of Fmoc-Gly and Fmoc-Trp derivatives, respectively, revealed that those amino acids do not have all of the characteristics for detection that were desired in this work. Fmoc-Lys(Boc), on the other hand, was shown to be the best derivatizing agent, due to the presence of Fmoc and Boc groups that allow the use of the combination of the UV, fluorescence and MS detection. One of its most prominent characteristics is the loss of 100 Da, cleaved during the ionization stage in the APCI source, which results in production of a relatively high signal. Owing to the range of structural possibilities presented by the various amino acids utilised, it is also possible to tune the chemical and chromatographic properties of the derivatives and the opportunity of charge inducing capabilities on deprotection36. Deprotection of the derivatives would increase their
49 The modified lipid extracts with Fmoc-Lys(Boc) allowed the identification of a greater number of sterols in an extract from a soil than previously characterized by GC-MS. The monitoring of a constant loss of 239 Da permitted the detection of only the derivatives and the mass spectra, obtained, after smoothing, are clean and straight forward to interpret. This method proved to be highly useful and suitable for a wide range of alcohols and sterols, independent of the presence of one or two hydroxyl functionalities and the molecular dimensions of the analyte. It would be very interesting to try the procedure developed in this work on GDGTs.
50
Appendix
In this paragraph the rest of mass spectra are shown.
Figure A-1 Mass spectrum of 7.
Figure A-2 Mass spectrum of 8.
Figure A-3 Mass spectrum of 9.
51 Figure A-5 Mass spectrum of 1,16-hexadecanolester Fmoc-Phe.
Figure A-6 Mass spectrum of unknown sterol m/z = 372.
Figure A-7 Mass spectrum of 1.
52 Figure A-9 Mass spectrum of 3.
Figure A-10 Mass spectrum of 4.
Figure A-11 Mass spectrum of 5.
53 Figure A-13 Mass spectrum of 7.
Figure A-14 Mass spectrum of 8.
Figure A-15 Mass spectrum of 9.
54 Figure A-17 Mass spectrum of 11.
Figure A-18 Mass spectrum of 12.
Figure A-19 Mass spectrum of 13.
55 Figure A-21 Mass spectrum of 1*.
Figure A-22 Mass spectrum of 2*.
Figure A-23 Mass spectrum of 3*.
