Synthesis and Characterization of Partially and Fully Saturated Menaquinone Derivatives

Menaquinones (MKs) contain both a redox active quinone moiety and a hydrophobic repeating isoprenyl side chain of varying lengths and degrees of saturation. This characteristic structure allows MKs to play a key role in the respiratory electron transport system of some prokaryotes by shuttling electrons and protons between membrane-bound protein complexes, which act as electron acceptors and donors. Hydrophobic MK molecules with partially and fully saturated isoprenyl side chains are found in a wide range of eubacteria and archaea, and the structural variations of the MK analogues are evolutionarily conserved but poorly understood. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, uses predominantly MK-9(II-H2) (saturated at the second isoprene unit) as its electron carrier and depends on the synthesis of MK-9(II-H2) for survival in host macrophages. Thus, MKs with partially saturated isoprenyl side chains may represent a novel virulence factor. Naturally occurring longer MKs are very hydrophobic, whereas MK analogues that have a truncated (i.e., one to three isoprenes) isoprenyl side chain are less hydrophobic. This improves their solubility in aqueous solutions, allowing rigorous study of their structure and biological activity. We present the synthesis and characterization of two partially saturated MK analogues, MK-2(II-H2) and MK-3(II-H2), and two novel fully saturated MK derivatives, MK-2(I,II-H4) and MK-3(I,II,III-H6).


■ INTRODUCTION
Menaquinones (or naphthoquinones, MKs) belong to the class of lipoquinones, which are a type of lipid-quinone containing isoprenoids that have a variety of biological roles. These molecules contain both a redox active quinone moiety and a hydrophobic repeating isoprenyl side chain of varying lengths and degrees of saturation ( Figure 1) depending on the organism. Although the naturally occurring lipoquinones generally have longer isoprenyl side chains, these MK analogues have very low water solubility, which causes problems for biological assays; therefore, the use of truncated MK derivatives can circumvent solubility issues in aqueous assays. The 1943 Nobel prize in physiology or medicine was awarded to Henrik Dam and Edward Doisy for the discovery and characterization of vitamin K (a MK), a key cofactor in blood coagulation. 1 The interest in and the importance of MK/vitamin K, its derivatives, and vitamers (e.g., vitamins K 1 , K 2 , and K 3 ) have remained strong. Vitamin K 1 , also known as phylloquinone, is found in green plants and contains four isoprene units with the terminal three saturated and functions as an electron acceptor during photosynthesis within photosystem I. 2 Vitamin K 2 , also known as menaquinone-4 (MK- 4), has been the subject of interesting reports, demonstrating its biological roles and applications over the last 10 years. 3−6 Vitamin K 3 (or menadione), the simplest 1,4-naphthoquinone derivative, which lacks the isoprenyl side chain and nonnaturally occurring N-alkylated MK derivatives has also shown interesting biological properties. 7−11 In 2017, the potent cyclic peptide antibiotic, Lotilibcin, was demonstrated to be effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus. 12 The activity of Lotilibcin was attributed to lysis of the bacterial membrane, where its membrane-disrupting effects depended on the presence of MK, which leaves the question if other MK derivatives have the same effect. 12 This report underlines the importance of understanding the relationship between the MK structure, its biological role, and its biological function.
MK analogues that contain partially and fully saturated isoprenyl side chains of various lengths are found in eubacteria and archaea. These characteristics are evolutionarily conserved and have long been used in taxonomic classification efforts and phenotypic organization of bacteria. 13−15 MK analogues with one or more saturated isoprene units are found in some members of Gram-positive and Gram-negative bacteria, and the systematic preparation and characterization of a few selected truncated partially and fully saturated MK derivatives are described in this manuscript. 14−17 In contrast to partial saturation, the fully saturated MK-7(14H) has been observed in archaea, which are single-celled microbes comprising part of the human microbiota. 18,19 It is thought that the full saturation of the isoprenyl side chain observed in archaea makes the cellular membrane more tolerant to extreme environments (e.g., high temperature, high salinity, and low/high pH). 18 Thus, many MK homologues that contain various degrees of saturation in their isoprenyl side chains have been isolated, but there is a limited understanding of the function of partially saturated derivatives. 16,20 Menaquinone-9(II-H 2 ) [abbreviated as MK-9(II-H 2 ), Figure 1A], which is saturated at the second isoprene, has been observed in a number of bacteria including Mycobacterium phlei and pathogenic Mycobacterium tuberculosis. 14,16,17,21,22 The biological significance of the partial saturation of MK-9 has remained unclear even 50 years after its first description. 20 Remarkably, the synthesis of MK-9(II-H 2 ) was recently shown to be essential for M. tuberculosis survival in host macrophages (white blood cells of the immune system); thus, MK-9(II-H 2 ) represents a potential novel virulence factor involved in tuberculosis (TB) disease progression. 23 Part of our ongoing studies includes why partial saturation of MK-9 increases the virulence of pathogens such as M. tuberculosis. 23 Therefore, we need access to MK analogues containing partially and fully saturated isoprenyl side chains. However, in general, they are neither commercially available nor sold as a pure geometric isomer as in the case of vitamin K 1 . The potential for the formation of cis and trans MK isomers and the separation and characterization of these geometric isomers are sometimes overlooked in the literature, but it is critical for their applications in biological systems. The use of cis/trans MK mixtures in biological systems would lead to inaccurate biological activities. Recently, the separation and nuclear magnetic resonance (NMR) analysis of cis/trans mixtures of the dietary supplement menaquinone-7 (MK-7) was reported, demonstrating the importance in understanding the different chemical, biochemical, conformational, and physical properties exhibited between cis and trans MK isomers. 24,25 Truncated MK analogues (i.e., one to three isoprene units) have the advantage of being less hydrophobic, which can improve their biological activity compared to longer isoprenyl MK analogues (e.g., MK-7 and MK-9) and can be prepared using a similar methodology described in this manuscript and reports elsewhere. 26,27 In this manuscript, we have synthesized and characterized four partially and fully saturated truncated isoprenyl MK derivatives ( Figure 1B−E) as well as separated and characterized the geometric isomers formed in the reactions. We have adopted the simple naming system where MK-2 with a saturated double bond at the second isoprene unit is denoted as menaquinone-2(II-H 2 ) and abbreviated as MK-2(II-H 2 ), where II denotes the second isoprene unit and H 2 denotes the saturation of one double bond ( Figure 1B). 23,28 This naming system follows for higher degrees of saturation such as MK-2(I,II-H 4 ), that is, saturated at isoprenes I and II and MK-3(I,II,III-H 6 ), that is, saturated at isoprenes I, II, and III ( Figure 1C,E). Although MK-2(II-H 2 ) and menaquinone-3(II-H 2 ) [abbreviated as MK-3(II-H 2 )] were known in literature reports mostly in the 1950s 29−34 and MK-2(II-H 2 ) was considered in a computational model study, 35 there are no modern syntheses reported, and these analogues have not been extensively characterized nor have their geometric isomers been resolved and characterized in the literature. Evidence supports the trans MK isomer as the only biologically active isomer. 27 Therefore, the separation and isolation of geometric isomers of MK derivatives is critical when testing these compounds in biological assays.
The synthesis and characterization of partially and fully saturated isoprenyl MK analogues, such as the two novel fully saturated MK derivatives described in this work, are important for understanding their requirement in biological systems, as little is known about their physical properties, reactivity, or 3Dconformation within solution or cell membranes. Access to these partially and fully saturated truncated MK analogues in the pure geometric form is critical, considering the recent discovery and timeliness for understanding why partially saturated MKs [e.g., MK-9(II-H 2 ), Figure 1A] may be a potential virulence factor against pathogenic M. tuberculosis, when currently around one-third of the world's population is infected with latent TB and over 1.3 million people worldwide die annually from TB. 36

■ RESULTS AND DISCUSSION
In the following, we describe the synthesis and characterization of two partially saturated MK analogues, MK-2(II-H 2 ) and MK-3(II-H 2 ), using modified and adapted procedures. We also describe the synthesis and characterization of two novel fully saturated MK analogues, MK-2(I,II-H 4 ) and MK-3(I,II,III-

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Article H 6 ), utilizing a key radical alkylation reaction. This is the first report on a preparatory scale utilizing a primary alkyl radical coupling reaction to access fully saturated MK derivatives, although in situ analysis of menaquinone-1(H 2 ) was reported previously. 37 Importantly, a convenient analysis can be carried out by rapid detection using benzene-d 6 as an NMR solvent for the evaluation of cis and trans MK mixtures (peaks coalesce in CDCl 3 disguising potential isomers formed). The cis/trans isomers can then be readily separated using preparative thinlayer chromatography (TLC) and analyzed.
Synthesis and Characterization of MK-2(II-H 2 ). The partially saturated MK analogue, MK-2(II-H 2 ) 3, was synthesized in a single step using Friedel−Crafts coupling between menadiol 1 and isophytol 2 (Scheme 1). The precursor menadiol was synthesized, as previously described, from menadione. 26,38 The partly hydroxylated heterogeneous Lewis−Brønsted acid catalyst, MgF 2 -48, was synthesized as previously described. 39−41 We chose MgF 2 -48 over other catalysts because it was readily prepared from inexpensive methanol and Mg 0 powder as well as from commonly available 48% aqueous hydrofluoric acid. 39−41 Furthermore, the partly hydroxylated MgF 2 catalyst was recently used with a biphasic n-heptane/polyene carbonate solvent system to synthesize vitamin K 1 and K 1 -chromanol. 40 Therefore, we thought this catalyst would be an excellent choice to synthesize the partially saturated MK-2(II-H 2 ), which has the analogous (but truncated) structure of vitamin K 1 [or MK-4(II,III,IV-H 6 )].
The Friedel−Crafts alkylation of 1 and 2 (Scheme 1) followed by in situ oxidation of dihydro-MK-2(II-H 2 ) formed the desired product 3 in an 11% yield. The low yield may be attributed to the formation of the undesired C2 isomeric side product (see Scheme 1), which was readily separated using column chromatography. The inherent instability of menadiol, which is prone to auto-oxidation even when purified immediately before use may also contribute to the low yield. MK-2(II-H 2 ) 3 was obtained as a yellow/red oil. The reaction was carried out in the dark, and the product was exposed minimally to light as previous studies have shown that interconversion to undesirable geometric isomers can occur through photo-oxidation if exposed to light. 16,42,43 We considered other possible catalysts to carry out the transformation shown in Scheme 1. For instance, we also explored a commercially available strong Brønsted acid catalyst (Nafion perfluorinated powdered resin and Nafion perfluorinated ion-exchange resin in 10 wt % dispersion in water); however, the yields were significantly lower with the increased formation of the C2 prenylated isomeric side product (see Scheme 1). Furthermore, we were concerned about the Nafion catalyst (containing super acidic sites) dehydrating isophytol to

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Article form undesirable phytadienes, 40 which presumably contribute to it being a less desirable catalyst for this purpose.
An interesting feature of MKs is the possibility of geometric isomer formation (Figure 2A,B). The biologically active trans isomer of vitamin K 1 is the natural biological form of vitamin K 1 . 27 Evidence also demonstrates that biological MKs have an all-trans configuration of the double bonds within the isoprenyl side chain. 27 The geometric isomers of vitamin K 1 , MK-7 and MK-9(II-H 2 ) (isolated from M. phlei, Figure 1A), have been successfully separated; therefore, efforts to isolate the trans isomer of MK-2(II-H 2 ) from its cis isomer byproduct ( Figure  2B) were carried out. 16,24,27 MK-2(II-H 2 ) 3 was initially isolated as a 2.7:1 mixture of trans/cis isomers (by NMR integration of alkene peaks in benzene-d 6 ) after column chromatography. The trans isomer of MK-2(II-H 2 ) 3 can be isolated using preparative TLC, similar to reports for vitamin K 1 analogues 44 and MK analogues. 16 The successful separation of the trans isomer of 3 by preparative TLC allowed for characterization of a single pure geometric isomer of MK-2(II-H 2 ), Figure 2. The structural assignment of trans-MK-2(II-H 2 ) ( Figure 2) was elucidated using two-dimensional (2D) NMR spectroscopic methods [ 1 H− 1 H correlation spectroscopy (COSY) and 1 H− 13 C heteronuclear single quantum correlation (HSQC) spectroscopy; see the Supporting Information for spectra, Figures S1− S4.1 and S7−S8.1]. Spectra obtained in benzene-d 6 (C 6 D 6 ) result in less overlap of the NMR signals for the cis/trans isomer mixture ( Figure 2C,D), compared to CDCl 3 where some of the geometric isomer peaks overlap (e.g., cis/trans alkene protons coalesce in CDCl 3 ) and the formation of isomers is not obvious. Therefore, the use of C 6 D 6 as a diagnostic NMR solvent for the evaluation of cis/trans mixtures for MK analogues was critical and is in line with a recently reported NMR analysis of cis/trans isomer mixtures of MK-7 24 (see the Supporting Information for spectra in CDCl 3 versus C 6 D 6 , Figure S40).
Synthesis and Characterization of MK-2(I,II-H 4 ). The fully saturated isoprenyl MK analogue, MK-2(I,II-H 4 ) 7, was synthesized in three steps from commercially available 3,7dimethyloctan-1-ol 4 (Scheme 2). The first step converted aliphatic alcohol 4 to the mesylate, which was then subjected to an S N 2 reaction using NaI to yield 1-iodo-3,7-dimethyloctane 5 in 47% yield over two steps. 38 The alkyl iodide 5 was then coupled to menadione 6 via radical alkylation using benzoyl peroxide 37 as a radical initiator to yield MK-2(I,II-H 4 ) 7 in 17% yield. The yield for the coupling reaction is close in range with that reported in the literature for other coupling reactions for the synthesis of MK analogues. 26,27,38 The unreacted alkyl iodide 5 can be recovered during column chromatography. In an effort to ensure selectivity of prenylation at C3 versus C2 (Scheme 2), the slow addition of napthoquinone 6 during the reaction was carried out so that the naphthoquinone stationary concentration in the reaction mixture is low compared to the prenyl iodide concentration. 37 MK-2(I,II-H 4 ) 7 was obtained as a yellow oil. This is the first report of the synthesis and characterization of MK-2(I,II-H 4 ) 7 (see the Supporting Information for the 2D NMR spectra, Figures S13−S14.1), the fully saturated analogue of MK-2.   33 First, the hydroxyl group of β-citronellol 8 was converted into iodide 9 through methane sulfonate in 66% yield over two steps. 38 The introduction of a carbonyl group to produce citronellylacetone 10 was accomplished under basic conditions in 88% yield over two steps. 45 This was followed by a Horner−Wadsworth− Emmons reaction to give methyl ester 11 in 62% yield. 38 Reduction of 11 with the Vitride reagent produced 6,7dihydrofarnesol 12 in 83% yield. 38,46 Finally, MK-3(II-H 2 ) 13 was obtained from the BF 3 -catalyzed coupling between the partially saturated allylic alcohol 12 and menadiol 1 (prepared from menadione) 26,38 in 12% yield. 26,38 The low yield may be attributed to the formation of the undesired C2 isomeric side product (see Scheme 3), which was readily separated using column chromatography. MK-3(II-H 2 ) 13 was obtained as a yellow oil.
Similar to MK-2(II-H 2 ) 3, MK-3(II-H 2 ) 13 was initially isolated as a 2.7:1 mixture (by NMR integration of alkene peaks in C 6 D 6 ) of trans/cis isomers. However, the trans isomer ( Figure 3A) can be isolated from the cis isomer byproduct ( Figure 3B) using preparative TLC, similar to a report on cis/ trans vitamin K 1 analogues. 44 MK analogues can be very sensitive; for example, during purification, we observed degradation of trans-MK-3(II-H 2 ) ( Figure 3A) within a solution of dichloromethane (DCM) that was not stabilized with amylene. This was evidenced by a decreased integration value for the alkene proton at 5.19 ppm and the appearance of two extra peaks at ∼1.3 ppm and a peak at 1.12 ppm in C 6 D 6 . Amylene is a radical scavenger, and it is likely that the alkene is undergoing a radical reaction with chloride radicals present in the unstabilized DCM. Similar to MK-2(II-H 2 ) 3, the use of benzene-d 6 as a diagnostic NMR solvent for the evaluation of cis/trans mixtures of MK-3(II-H 2 ) 13 ( Figure 3C,D) was essential because the presence of the cis isomer was not wellobserved in CDCl 3 (see the Supporting Information for spectra in CDCl 3 vs benzene-d 6 , Figure S40).
The structural assignment of trans-MK-3(II-H 2 ) ( Figure 3) was elucidated using 2D NMR spectroscopic methods [ 1 H− 1 H COSY, 1 H− 13 C HSQC, and 1 H− 1 H rotating frame Overhauser effect spectroscopy (ROESY); see the Supporting Information for spectra, Figures S23−S26.1 and S29−S31]. The assignment of the alkene proton H h at 5.13 ppm was determined by the following: the observation of a cross peak in the COSY spectrum at 5.13 and 1.73 ppm (assignment H w , Figure 3), the observation of a cross peak at 5.13 and 1.94 ppm (assignment H n , Figure 3), and the lack of an observed cross peak at 5.19 and 1.73 ppm (H w ). The assignment of alkene proton H i at 5.19 ppm was determined by the observation of a cross peak at 5.19 and 1.67 ppm and the lack of a cross peak at 5.19 and 1.73 ppm (H w ). 1 H− 1 H 2D ROESY NMR spectroscopy (see the Supporting Information) also supported the alkene assignment by the observation of a ROE cross peak at 5.19 and 1.67 ppm (H i −H z interaction) and the lack of a ROE cross peak at 5.19 ppm and 1.56 ppm. The assignment of the terminal methyl groups, H y and H z , was also determined by 1 H− 1 H 2D ROESY NMR spectroscopy. It is anticipated that a ROE cross peak would be observed between the alkene proton H i and the methyl group, that is, cis to H i , because of the close spatial proximity, and a ROE cross peak is observed, as expected, at 5.19 and 1.67 ppm (H i −H z interaction) (see the Supporting Information). Although H y and H i are within the spatial limits of an expected ROE cross peak observation, an ROE cross peak is not observed at 5.19 ppm and 1.56 ppm. This supports

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Article the structural assignment of H y as trans to the alkene proton H i , as H y is further away, spatially, from H i compared to H z .
Synthesis and Characterization of MK-3(I,II,III-H 6 ). The fully saturated isoprenyl MK analogue, MK-3(I,II,III-H 6 ) 17, was synthesized in four steps (Scheme 4) from the commercially available allylic alcohol, farnesol 14. The first step was the Pd-catalyzed transfer hydrogenation of farnesol to yield the fully saturated alcohol 15 in 88% yield. 47 Subsequent mesylation of the alcohol of product 15 followed by substitution with NaI gave aliphatic iodide 16 in 47% yield over two steps. 38,48 The final step was the radical coupling of menadione 6 and aliphatic iodide 16 using benzoyl peroxide 37 as the radical initiator to yield the desired final product, MK-3(I,II,III-H 6 ) 17, in 15% yield. This yield is similar to that attained for the coupling reaction to obtain MK-2(I,II-H 4 ) 7.
The unreacted aliphatic iodide 16 can be recovered during purification. Similar to MK-2(I,II-H 4 ) 7, in an attempt to ensure good selectivity of prenylation (C3 over C2, Scheme 4), slow addition of napthoquinone 6 to the prenyl iodide solution during the course of the reaction was carried out. 37 Similar to the synthesis of MK-2(I,II-H 4 ) 7 above, this is the second report of this type of primary alkyl radical coupling reaction to access MK and the first report on a preparatory scale. MK-3(I,II,III-H 6 ) 17 was obtained as a yellow oil. This is the first report for the synthesis and characterization of MK-3(I,II,III-H 6 ) 17 (see the Supporting Information for 2D NMR spectra, Figures S38−S39.1), the fully saturated analogue of MK-3.

■ CONCLUSIONS
The results of these studies will allow for the synthesis of various MK analogues and give access to previously unexplored compounds with unknown biological activity. In summary, this manuscript described the synthesis and characterization of four truncated partially and fully saturated isoprenyl MK analogues as well as separated and characterized the geometric isomers formed in the reactions. The partially saturated MK-2(II-H 2 ) was successfully synthesized in a single step from menadiol and isophytol, and the trans isomer was separated from the cis isomer byproduct by preparative TLC, followed by characterization. The novel fully saturated MK-2(I,II-H 4 ) was successfully synthesized in three steps from 3,7-dimethyloctan-1-ol. The partially saturated MK-3(II-H 2 ) was successfully synthesized in seven steps from β-citronellol, and the trans isomer was readily separated from the cis isomer byproduct by preparative TLC, followed by characterization. Finally, the novel fully saturated MK-3(I,II,III-H 6 ) was successfully synthesized in four steps from farnesol. The use of benzened 6 as a diagnostic NMR solvent for the rapid evaluation of cis/ trans mixtures for MK/vitamin K analogues was essential as cis/trans peaks often coalesce in chloroform-d.
The separation and characterization of an MK analogue's trans isomer is essential for use of these compounds in biological systems or for understanding biological systems that use MK because evidence shows that the cis isomer is not biologically active. The partially and fully saturated MK analogues synthesized in this manuscript are substrate analogues for enzymes involved in MK/vitamin K metabolism and are essential for understanding the structure−activity relationships of these biological systems. 49 They are also critical for understanding the function, reactivity, and conformation of partially saturated MK analogues as little is currently known. These MK analogues are also of interest from a therapeutic and medicinal standpoint. For instance, MK-4 (vitamin K 2 ) was recently shown to be a potential new therapeutic strategy to treat rheumatoid arthritis; it is a proposed drug for the effective treatment of osteoporosis and has anticancer properties. Nevertheless, the synthesis of partially saturated MK analogues remains a challenge as there is currently not a suitable catalyst to selectively saturate an individual isoprene unit on an MK analogue while leaving the other double bonds unaffected. Importantly, the synthesis and characterization of these partially and fully saturated MK substrate analogues is essential for understanding why they are necessary for various organisms to survive, and most importantly, understanding the reason MK-9(II-H 2 ) (i.e., saturated at the second isoprene unit) is a potential virulence factor for pathogenic M. tuberculosis, which is responsible for the deaths of ∼1.3 million people annually.

■ EXPERIMENTAL SECTION
General Information. Menadiol 1 was synthesized as previously described 26,38 from menadione, and the MgF 2 -48 sol−gel catalyst was synthesized, as previously described. 39−41 For clarity, any mention of MK-2(II-H 2 ) or MK-3(II-H 2 ) is assumed to be the cis/trans mixture, whereas the trans isomer is specifically written as trans-MK-2(II-H 2 ) or trans-MK-3(II-H 2 ). We used the standard abbreviations adopted by the American Chemical Society, for example, ethyl acetate
NMR Spectroscopic Studies. The synthesized compounds were dried under vacuum for ∼2−4 days (∼150− 200 mTorr), unless otherwise noted, to ensure as much residual solvent from purification was removed as possible. Samples for NMR studies were prepared immediately prior to running the samples using deuterated solvents sealed under an inert atmosphere in glass ampules. The NMR sample solutions were placed under an argon atmosphere immediately and capped, and then the spectra were acquired, unless otherwise noted. NMR samples of MKs were protected from light by wrapping the samples in an aluminum foil while not in the NMR instrument because of potential degradation. 1 H and 13 C spectra were recorded using a Varian model MR400 or a Bruker model AVANCE Neo400 spectrometer equipped with a BBFO smart probe and an automated tuning module operating at 400 or 101 MHz, respectively. Chemical shift values (δ) are reported in ppm and referenced against the internal solvent peaks in 1 H NMR (CDCl 3 , δ at 7.26 ppm; benzene-d 6 , δ at 7.16 ppm) and in 13  Preparation of MK-2(II-H 2 ) (3). Compound 3 was accessed from 1 and 2.
Preparation of 2-(3,7-Dimethyloct-2-en-1-yl)-3-methylnaphthalene-1,4-dione MK-2(II-H 2 ) (3). To a dry 150 mL round-bottom thick-walled pressure flask was added a dry stir bar, dry heptane (21 mL), and polyene carbonate (21 mL). The solution was bubbled with argon for several min. Then, menadiol 1 (1.22 g, 7.00 mmol) was added and stirred until dissolution occurred under argon bubbling. Then, isophytol 2 (1.09 g, 7.00 mmol) was added, followed by MgF 2 -48 (350 mg). The biphasic reaction mixture was bubbled with argon for ∼1 min and then sealed tightly with a Teflon screw cap. The sealed reaction mixture was covered in an aluminum foil to minimize light exposure and was stirred for 5 h at 100°C under an argon atmosphere. The yellow biphasic suspension was then cooled to an ambient temperature. The 1 H NMR spectrum of the crude product confirmed product formation by the presence of a doublet at 3.37 ppm (CDCl 3 ). The layers were separated, and the polyene carbonate layer (bottom layer) was extracted with n-pentane (3 × 100 mL). The combined yellow organic extracts were washed with DDI H 2 O (100 mL), washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure at an ambient temperature. The crude oil was dried overnight under reduced pressure to yield 1.31 g of crude red oil. The crude red oil was purified by flash column chromatography (300 mL of 230− 400 mesh SiO 2 , 20:1 n-pentane/EtOAc, 40 mm column) to yield 0.235 g (0.757 mmol, 10.8% yield) as a yellow/red oil after drying under vacuum (200 mTorr). The C2 isomeric side product was readily separated by flash column chromatography eluting after the C3 desired product. The product was obtained as a mixture of trans/cis isomers (ratio of trans/cis = 2.7:1 by NMR integration of alkene peaks), as has similarly been reported for vitamin K 1 analogues 44 and various MK analogues. 27   A sample for analytical characterization of trans-MK-2(II-H 2 ) was purified by normal phase preparative TLC (see experimental below) on silica gel using (30:1) n-pentane/ EtOAc as the eluting solvent; R f cis: 0.37, R f trans: 0.32; ratio of trans/cis before separation was 2.7:1 (the ratio was measured from the NMR integration of alkene peaks). Although we isolated the pure trans isomer, the cis isomer coeluted with the trans isomer, and efforts to obtain a sample of the pure cis isomer were not successful. The cis and the trans isomers have different NMR spectra in C 6 D 6 for the protons and carbons within the isoprenyl side chain. Data for trans-MK-2(II-H 2 ): 1 13  A sample for analytical characterization of trans-MK-2(II-H 2 ) was prepared by using normal phase preparative TLC. First, 8.0 mg of cis/trans-MK-2(II-H 2 ) (dissolved in a minimal amount of DCM) was loaded onto a preparative TLC plate via a capillary tube and then eluted once using 30:1 n-pentane/ EtOAc and an elution time of 45 min. The TLC plate was

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Article briefly air-dried and then eluted a second time under the same conditions. The yellow band was illuminated under ultraviolet light, and while illuminated, the band was divided into a top two-third and a bottom one-third. The bottom one-third was carefully removed with a razor blade, extracted with DCM, filtered through a disposable Pasteur pipette filled with glass wool (prerinsed with DCM), concentrated under reduced pressure at an ambient temperature, and dried for 4 days at reduced pressure (200 mTorr) to provide 2.5 mg of trans-MK-2(II-H 2 ) as a yellow oil (31.3% recovery).
1-Iodo-3,7-dimethyloctane (5). To a dry 250 mL roundbottom Schlenk flask was added a dry stir bar, 3,7-dimethyl-1octanol 4 (8.61 g, 54.4 mmol), and dry DCM (100 mL), which was then purged/evacuated with argon repeatedly. The solution was cooled to 0°C in an ice−H 2 O bath. Then, methanesulfonyl chloride (2.16 mL, 3.20 g, 27.9 mmol, 1.2 equiv) was added under an argon atmosphere followed by dropwise addition of dry triethylamine (11.39 mL, 8.26 g, 81.6 mmol, 1.5 equiv) over 5 min at 0°C. The resulting solution was stirred at an ambient temperature for 1.5 h under argon. The reaction was quenched with sat. NaHCO 3 (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , and then concentrated under reduced pressure at an ambient temperature (residual triethylamine can be removed by vacuum pumping for ∼1 h) to yield 9.65 g of crude yellow oil. The crude mesylate was dissolved in dry and degassed acetone (100 mL, dried over anhydrous K 2 CO 3 immediately prior for ∼10 min) in a dry 250 mL round-bottom Schlenk flask. Then, NaI (16.50 g, 110.0 mmol, 2.0 equiv) was added, and the solution was refluxed for 2 h under argon. The white reaction mixture was diluted with ice−DDI H 2 O (100 mL) and diethyl ether (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , and then concentrated under reduced pressure at an ambient temperature to yield 9.83 g of crude clear and yellow oil. The crude product was purified by flash column chromatography (800 mL of 230−400 mesh SiO 2 , 10:1 n-pentane/EtOAc, 70 mm column) to yield 6 2-(3,7-Dimethyloctyl)-3-methylnaphthalene-1,4-dione MK-2(I,II-H 4 ) (7). To a dry 100 mL round-bottom Schlenk flask was added a dry stir bar and dry and degassed benzene (30 mL) followed by 1-iodo-3,7-dimethyloctane 5 (2.68 g, 10.0 mmol, 1 equiv). To a dry 20 mL vial was added dry and degassed benzene (15 mL) followed by menadione 6 (1.72 g, 10.0 mmol) and benzoyl peroxide (2.42 g, 10.0 mmol, 1 equiv) and then sonicated until dissolution occurred under argon. The naphthoquinone−benzoyl peroxide solution was added drop-wise for over 2 h 15 min to the refluxing prenyl iodide solution, which was under an argon atmosphere during the reaction. After the addition was complete, the solution was refluxed for an additional 1 h. The solution was diluted with sat. NaHCO 3 (100 mL) and diethyl ether (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , and then concentrated under reduced pressure at an ambient temperature to yield a crude yellow powder. The crude powder was purified by flash column chromatography (10:1.5 n-pentane/EtOAc) to yield 0.539 g (1.73 mmol, 17.3% yield) after drying under vacuum (150 mTorr) as a yellow oil. 1  Preparation of MK-3(II-H 2 ) (13). Compound 13 was accessed through compounds 9, 10, 11, and 12 starting from commercially available compound 8.

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Article mL). The combined yellow organic extracts were washed with sat. NaHCO 3 (100 mL), washed with DDI H 2 O (100 mL), washed with brine (100 mL), dried with anhydrous Na 2 SO 4 , and then concentrated under reduced pressure at an ambient temperature to yield 5.06 g of the crude red solid. The crude solid was purified by flash column chromatography (1000 mL of 230−400 mesh SiO 2 , 20:1 n-pentane/EtOAc) to yield 1.28 g (yield at this stage = 29.4%, 3.38 mmol) as a red oil, but further purification was needed as evidenced by 1 H NMR analysis. The C2 isomeric side product was readily separated by flash column chromatography eluting after the C3 desired product. The red oil was subjected to a second column chromatography purification using the same conditions as the first purification, and then the material was dried under reduced pressure (150 mTorr) overnight to yield 0.501 g (1.32 mmol, 11.5% yield) as a yellow oil. Similar to vitamin K 1 analogues, 44 various MK analogues 27 and MK-2(II-H 2 ) 3, the product was obtained as a mixture of trans/cis isomers (ratio of trans/cis = 2.7:1 by NMR integration of alkene peaks). 1   A sample for analytical characterization of trans-MK-3(II-H 2 ) was prepared similarly as described (see experimental below) for trans-MK-2(II-H 2 ) above by normal phase preparative TLC on silica gel using (30:1) n-pentane/EtOAc as the eluting solvent; R f cis: 0.35, R f trans: 0.30; ratio of trans/ cis before separation was 2.7:1 (ratio by NMR integration of alkene peaks). Importantly, the bottom one-half of the band was isolated, instead of the bottom one-third of the band as was done to obtain trans-MK-2(II-H 2 ). This purification provided 3.7 mg of trans-MK-3(II-H 2 ) as a yellow oil (43.5% recovery) from the initial 8.5 mg cis/trans mixture loaded onto the TLC plate after drying under vacuum (200 mTorr) for 4 days. Similar to MK-2(II-H 2 ) 3, we isolated the pure trans isomer; however, the cis isomer coeluted with the trans isomer, and efforts to obtain a sample of the pure cis isomer were not successful. Similar to MK-2(II-H 2 ) 3, the cis and trans isomers of MK-3(II-H 2 ) have different NMR spectra in C 6 D 6 for the protons and carbons that belong to the isoprenyl side chain.  13  3,7,11-Trimethyldodecan-1-ol (15). To a dry 500 mL round-bottom Schlenk flask was added a dry stir bar, dry methanol (200 mL), ammonium formate (29.15 g, 462.3 mmol, 9.28 equiv), and farnesol 14 (11.08 g, 49.83 mmol), followed by careful addition of the palladium catalyst by a funnel (2.14 g, 20.1 mmol, 0.4 equiv). The reaction mixture was stirred at an ambient temperature for 5 days under positive pressure from an argon balloon, and 1 H NMR analysis showed only residual alkene present. The reaction mixture was filtered through a pad of Celite and washed with MeOH (600 mL). MeOH was removed under reduced pressure at an ambient temperature. The crude product was then filtered through a silica plug (4:1 n-pentane/EtOAc, 500 mL eluent) and washed with EtOAc (300 mL). The organic filtrate was concentrated under reduced pressure at an ambient temperature to yield a crude clear and colorless oil. The crude oil was suspended in diethyl ether (200 mL), washed with sat. NaHCO 3 (200 mL), washed with brine (100 mL), dried over anhydrous Na 2 SO 4 , and then concentrated under reduced pressure at an ambient temperature to yield a crude clear and colorless oil. The crude oil was purified by flash column chromatography (1000 mL of 230−400 mesh SiO 2 , 4:1 n-pentane/EtOAc, 70 mm column) to yield 9.99 g (43.7 mmol, 87.8% yield) as a clear and colorless oil after drying under vacuum (150 mTorr). 1  1-Iodo-3,7,11-trimethyldodecane (16). To a dry 250 mL round-bottom Schlenk flask was added a dry stir bar, 3,7,11trimethyldodecan-1-ol 15 (9.41 g, 41.2 mmol), and dry DCM (125 mL). The flask was purged/evacuated with argon repeatedly. The solution was cooled to 0°C in an ice−H 2 O bath. Then, dry triethylamine (8.61 mL, 6.25 g, 61.8 mmol, 1.5 equiv) was added to the flask followed by dropwise addition of methanesulfonyl chloride (3.82 mL, 5.66 g, 49.4 mmol, 1.2 equiv) at 0°C for over 5 min while stirring under argon. The reaction mixture was stirred at an ambient temperature for 40 min under argon. The off-white reaction mixture was quenched with sat. NaHCO 3 (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous MgSO 4 , vacuum-filtered, and then concentrated under reduced pressure at an ambient temperature (residual triethylamine can be removed by vacuum pumping for ∼1 h) to yield 12.98 g of crude clear and lightyellow oil. To another dry 250 mL round-bottom Schlenk flask

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Article was added dry and degassed acetone (100 mL, dried over anhydrous K 2 CO 3 for ∼10 min immediately prior) and crude mesylate followed by NaI (15.44 g, 103.0 mmol, 2.5 equiv). The reaction mixture turned bright yellow upon addition of NaI and was refluxed for 1 h and 40 min under argon. The yellow reaction mixture was diluted with diethyl ether (100 mL) and DDI H 2 O (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous MgSO 4 , vacuum-filtered, and then concentrated under reduced pressure at an ambient temperature to yield a crude yellow murky oil. To remove the salt impurities, the crude oil was suspended in n-pentane (100 mL) and washed with DDI H 2 O (100 mL), and then the phases were separated. The aqueous layer was extracted with npentane (3 × 100 mL). The combined organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous MgSO 4 , vacuum-filtered, and then concentrated under reduced pressure at an ambient temperature to yield 13.19 g of crude clear and light-yellow oil. The crude oil was purified by flash column chromatography (1000 mL of 230−400 mesh SiO 2 , 100% n-pentane, R f = 0.71, 70 mm column) to yield 6 2-Methyl-3-(3,7,11-trimethyldodecyl)naphthalene-1,4dione MK-3(I,II,III,H 6 ) (17). To a dry 100 mL round-bottom Schlenk flask was added under argon, a dry stir bar, dry and degassed benzene (30 mL), and 1-iodo-3,7,11-trimethyldodecane 16 (3.38 g, 10.0 mmol, 1.00 equiv). To a dry 20 mL vial was added benzene (15 mL), which was degassed via argon needle purge, followed by menadione 6 (1.72 g, 10.0 mmol, 1.00 equiv) and benzoyl peroxide (2.42 g, 10.0 mmol, 1.00 equiv) and then sonicated until dissolution occurred under argon. The naphthoquinone−benzoyl peroxide solution was added dropwise to the refluxing prenyl iodide solution for over 2 h and 15 min while the reaction mixture was refluxing under argon. The solution was then refluxed for an additional 1 h. The reaction mixture was diluted with sat. NaHCO 3 (100 mL) and DDI H 2 O (100 mL), and then the phases were separated. The aqueous layer was extracted with diethyl ether (3 × 100 mL). The combined yellow organic extracts were washed with sat. NaHCO 3 (100 mL), washed with brine (100 mL), dried over anhydrous MgSO 4 , and concentrated under reduced pressure at an ambient temperature to yield 6.63 g of crude yellow powder. The crude product was purified by flash column chromatography (1000 mL of 230−400 mesh SiO 2 , 10:1 n-pentane/EtOAc, 70 mm column) to yield 0.555 g (1.45 mmol, 14.5% yield) after drying under vacuum (200 mTorr) as a yellow oil. 1