catalysts

Review Polar Head Modified Phospholipids by Phospholipase D-Catalyzed Transformations of Natural Phosphatidylcholine for Targeted Applications: An Overview

Chiara Allegretti 1,* , Francesca Denuccio 2, Letizia Rossato 2 and Paola D’Arrigo 1,3,*

1 Department of Chemistry, Materials and Chemical Engineering “Giulio Natta”, Politecnico di Milano, via Mancinelli 7, 20131 Milano, Italy 2 Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 Milano, Italy; [email protected] (F.D.); [email protected] (L.R.) 3 Consiglio Nazionale delle Ricerche (C.N.R.), Istituto di Scienze e Tecnologie Chimiche “Giulio Natta”, SCITEC, via Mancinelli 7, 20131 Milano, Italy * Correspondence: [email protected] (C.A.); [email protected] (P.D.)



Received: 28 July 2020; Accepted: 13 August 2020; Published: 1 September 2020


Abstract: This review describes the use of phospholipase D (PLD) to perform the transphosphatidylation of the most common natural phospholipid (PL), phosphatidylcholine (PC) to obtain polar head modified phospholipids with real targeted applications. The introduction of different polar heads with distinctive physical and chemical properties such as charge, polarity and dimensions allows the obtainment of very different PLs, which can be exploited in very diverse fields of application. Moreover, the inclusions of a bioactive moiety in the PL polar head constitutes a powerful tool for the stabilization and administration of active ingredients. The use of this biocatalytic approach allows the preparation of compounds which cannot be easily obtained by classical chemical methods, by using mild and green reaction conditions. PLD is a very versatile enzyme, able to catalyze both the hydrolysis of PC to choline and phosphatidic acid (PA), and the transphosphatidylation reaction in the presence of an appropriate alcohol. The yield of production of the desired product and the ratio with the collateral PA formation is highly dependent on parameters such as the nature and concentration of the alcohol and the enzymatic source. The application of PLD catalyzed transformations for the production of a great number of PLs with important uses in medical, nutraceutical and cosmetic sectors will be discussed in this work.

Keywords: phospholipase D; phospholipids; transphosphatidylation

1. Introduction Phospholipids (PLs) are amphiphilic molecules that are ubiquitous in nature and play a fundamental structural role in all biological membranes [1–3]. These molecules present a glycerol backbone which is linked to two fatty acid-derived acyl residues at the carbon positions sn-1, sn-2 and to a phosphodiester group bearing a polar head at the sn-3 position, as reported in Figure1. PL molecules, therefore, present a hydrophobic moiety comprising the two fatty acid chains which may be either saturated or unsaturated, the glycerol backbone of intermediate polarity and a polar hydrophilic head group. Most of the different classes of PLs are defined by the nature of the polar head, but their structural diversity can also be linked to the type of the fatty acid substituents and their regioisomerism. PLs are widely studied and investigated as they are biologically active compounds. Besides being the major component of all biological membranes, they present excellent biocompatibility and have very interesting applications by virtue of the intrinsic properties of the individual molecules

Catalysts 2020, 10, 997; doi:10.3390/catal10090997 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 997 2 of 19 themselves. They are a nutritious source of fatty acid, organic phosphate and choline, and they are recognized to possess beneficial effects to human health [4,5]. For these reasons, they are the topic of many areas of biomedical research, and they can generate model systems of biological membranes for specific studies [6]. In fact, they are implicated in many cellular functions. They are involved in processes of cellular regeneration and differentiation in neurological pathways; they are able to promote the biological activity of various membrane linked proteins and receptors; they play an important part for molecule transportation through the cell membrane; and they can be used as diagnostic markers for certain diseases and therapeutic agents [7]. Dietary PLs are essential for preventing a range of human diseases such as coronary heart disease, cancer and inflammation [8,9]. Furthermore, the amphiphilic nature of these molecules is the key factor responsible for their unique property of spontaneous aggregation in aqueous environments. They are able to self-associate spontaneously forming supramolecular structures such as bilayers, micelles and liposomes, and this characteristic is very appealing for the drug delivery, diagnostic testing and cosmetic sectors [10–14]. The PL based drug delivery system has been found to provide an effective delivery of drugs, and a great variety of PLs formulations has been used for clinical applications [15,16]. In particular, the gastro-resistance of liposomes is the key factor in the formulation of bioactive molecules: vitamins, antioxidants, essential oils, flavors, enzymes and fatty acids are some examples of products encapsulated therein. Additionally PLs possess considerable industrial interest as emulsifiers, food stabilizers, antioxidants, detergents and surfactants [17–19]. All these applications lead to a strong demand for PL preparation methodologies [5,15,20,21]. Catalysts 2020, 10, x FOR PEER REVIEW 2 of 19

Figure 1. 1. StructureStructure of the of themain main natural natural phospholipids phospholipids (PLs) (PLs)(PC: (PC:phosphatidylcholine; phosphatidylcholine; PG: phosphatidylglycerol;PG: phosphatidylglycerol; PE: PE: phosphatidylethanolamine; phosphatidylethanolamine; PS: phosphatidylserine; PS: phosphatidylserine; PI: phosphatidylinositol). PI: phosphatidylinositol). PLs are widely distributed in nature and can be obtained from animal products such as milk, egg yolk,PL molecules krill, bovine, therefore brain and, present from a vegetable hydrophobic sources moiety such comprising as soybeans, the sunflower, two fatty corn, acid peanut,chains whichflaxseed, may rape be either (canola) saturated seed and or unsaturated, wheatgerm [ 22the,23 glycerol]. The termbackbone lecithin of intermediate refers to a mixture polarity ofand PLs a polarextracted hydrophilic both from head animal group. and vegetableMost of the sources different which classes provide of aPLs diff aerentre defined composition by the of nature PLs in of terms the polarof polar head heads, but and their acyl structural chains. Nowadays, diversity can lecithins also be are linked typically to the extracted type of industriallythe fatty acid by substituents treatment of andvegetable their regioisomerism. oils, soy beans, eggPLs yolk, are widely milk, biomass studied andand animalinvestigated organs as [22 they]. The are principle biologically sources active in compounds.terms of quantity Besides for commercialbeing the major lecithins component are egg of yolk all andbiological soybeans. membranes, Lecithins they are mainlypresent composed excellent biocompatibilityby PLs, triglycerides, and have carbohydrates very interesting and minor applications components by virtue [24]. of The the most intrinsic common properties PLs present of the individualin lecithins molecules are phosphatidylcholine themselves. They (PC), are phosphatidylethanolamine a nutritious source of fatty (PE) acid, and organic phosphatidylinositol phosphate and (PI).choline The, and commercial they are recognized value of PLs to inpossess the pharmaceutical, beneficial effects cosmetic to human and health food industry[4,5]. For increasesthese reasons, with theythe purity are the of topic the product,of many whichareas of refers biomedical to the nature research of, polarand they head can and generate lipophilic model tails systems which are of biologicalfundamental membranes for obtaining for specific the desired studies functions [6]. In [fact,25,26 they]. In are fact, implicated the chemical in many and physical cellular propertiesfunctions. Theyof PLs are depend involved on theirin processes molecular of cellular structure regeneration in terms of and the differentiation nature of the polar in neurological head group pathways; and fatty theyacid composition,are able to promote which is the highly biological linked activity to their origin.of various For membrane natural PLs, linked it is well proteins known and that receptors the purer; they play are, the an higherimportant the part price. for Partial molecule purification transportation of PLs fromthrough lecithins the cell is possiblemembrane after; and many they steps can be of used as diagnostic markers for certain diseases and therapeutic agents [7]. Dietary PLs are essential for preventing a range of human diseases such as coronary heart disease, cancer and inflammation [8,9]. Furthermore, the amphiphilic nature of these molecules is the key factor responsible for their unique property of spontaneous aggregation in aqueous environments. They are able to self-associate spontaneously forming supramolecular structures such as bilayers, micelles and liposomes, and this characteristic is very appealing for the drug delivery, diagnostic testing and cosmetic sectors [10–14]. The PL based drug delivery system has been found to provide an effective delivery of drugs, and a great variety of PLs formulations has been used for clinical applications [15,16]. In particular, the gastro-resistance of liposomes is the key factor in the formulation of bioactive molecules: vitamins, antioxidants, essential oils, flavors, enzymes and fatty acids are some examples of products encapsulated therein. Additionally PLs possess considerable industrial interest as emulsifiers, food stabilizers, antioxidants, detergents and surfactants [17–19]. All these applications lead to a strong demand for PL preparation methodologies [5,15,20,21]. PLs are widely distributed in nature and can be obtained from animal products such as milk, egg yolk, krill, bovine brain and from vegetable sources such as soybeans, sunflower, corn, peanut, flaxseed, rape (canola) seed and wheatgerm [22,23]. The term lecithin refers to a mixture of PLs extracted both from animal and vegetable sources which provide a different composition of PLs in terms of polar heads and acyl chains. Nowadays, lecithins are typically extracted industrially by treatment of vegetable oils, soy beans, egg yolk, milk, biomass and animal organs [22]. The principle sources in terms of quantity for commercial lecithins are egg yolk and soybeans. Lecithins are mainly composed by PLs, triglycerides, carbohydrates and minor components [24]. The most common PLs present in lecithins are phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI). The commercial value of PLs in the pharmaceutical, cosmetic and food

Catalysts 2020, 10, x FOR PEER REVIEW 3 of 19 industry increases with the purity of the product, which refers to the nature of polar head and lipophilic tails which are fundamental for obtaining the desired functions [25,26]. In fact, the chemical and physical properties of PLs depend on their molecular structure in terms of the nature of the polar headCatalysts group2020, 10and, 997 fatty acid composition, which is highly linked to their origin. For natural PLs,3 it of is 19 well known that the purer they are, the higher the price. Partial purification of PLs from lecithins is possible after many steps of solvent partitioning and chromatographic separations. In this way, PC, solvent partitioning and chromatographic separations. In this way, PC, the most abundant natural PL, the most abundant natural PL, can be isolated and purified. However, the obtainment of other specific can be isolated and purified. However, the obtainment of other specific minor PLs that rarely exist minor PLs that rarely exist in nature is quite difficult and suffers the high cost of chromatographic in nature is quite difficult and suffers the high cost of chromatographic separation, which makes it separation, which makes it inappropriate for the purification of PLs in industrial processes. inappropriate for the purification of PLs in industrial processes. The preparation of well-defined and novel PLs is also at the base of the investigation of their The preparation of well-defined and novel PLs is also at the base of the investigation of their potential properties. There is a great interest in producing a wide variety of innovative PLs with potential properties. There is a great interest in producing a wide variety of innovative PLs with modulated chemical and physical properties, such as charge, size, polarity, type of polar head or side modulated chemical and physical properties, such as charge, size, polarity, type of polar head or side chains, for diverse uses. Some applications require, in fact, the preparation of PLs with specific polar chains, for diverse uses. Some applications require, in fact, the preparation of PLs with specific polar heads and different and defined acyl chains at the sn-1 and sn-2 positions [27,28]. The preparation of heads and different and defined acyl chains at the sn-1 and sn-2 positions [27,28]. The preparation these products is challenging and can be obtained by total synthesis from appropriate homochiral of these products is challenging and can be obtained by total synthesis from appropriate homochiral precursors (such as glycerol derivatives) by performing a multistep synthesis which generally precursors (such as glycerol derivatives) by performing a multistep synthesis which generally requires requires the use of a complex sequence of protection and deprotection steps in order to introduce the the use of a complex sequence of protection and deprotection steps in order to introduce the desired desired functional groups [29–32]; however, this aspect is beyond the focus of this review. A functional groups [29–32]; however, this aspect is beyond the focus of this review. A biocatalytic biocatalytic approach aiming to synthesize the modified PLs is preferred if compared to the chemical approach aiming to synthesize the modified PLs is preferred if compared to the chemical synthesis synthesis especially if the product’s destination is for the food or pharmaceutical industry because of especially if the product’s destination is for the food or pharmaceutical industry because of the use the use of harmless, natural and much safer reagents [33–35]. The use of enzymes offers a higher of harmless, natural and much safer reagents [33–35]. The use of enzymes offers a higher selectivity; selectivity; it allows the use of milder conditions by avoiding the use of strong, unsafe and often it allows the use of milder conditions by avoiding the use of strong, unsafe and often corrosive reagents; corrosive reagents; and permits the obtainment of products which cannot easily be achieved by and permits the obtainment of products which cannot easily be achieved by classical chemical methods. classical chemical methods. PLs have two carboxylic and two phosphate ester bonds: their enzyme-mediated modification PLs have two carboxylic and two phosphate ester bonds: their enzyme-mediated modification includes hydrolysis, transphosphatidylation and transesterification. The enzymes involved in these includes hydrolysis, transphosphatidylation and transesterification. The enzymes involved in these transformations are called phospholipases and are selective for one of the four ester bonds present in transformations are called phospholipases and are selective for one of the four ester bonds present in the molecules of PLs, as described in Figure2 for natural PC (reported as 1-palmitoyl-2-linoleyl-PC). the molecules of PLs, as described in Figure 2 for natural PC (reported as 1-palmitoyl-2-linoleyl-PC). Phospholipases A1 (PLA1, 3.1.1.32)/A2 (PLA2, 3.1.1.4) remove, respectively, the fatty acids in Phospholipases A1 (PLA1, 3.1.1.32)/A2 (PLA2, 3.1.1.4) remove, respectively, the fatty acids in position position sn-1 and sn-2 producing 2-acyl and 1-acyl-lysophospholipids (1-lysoPL and 2-lysoPL) [29]. sn-1 and sn-2 producing 2-acyl and 1-acyl-lysophospholipids (1-lysoPL and 2-lysoPL) [29]. Phospholipase C C (PLC, (PLC, E.C.3.1.4.3) E.C.3.1.4.3) cleaves cleaves the the phosphoric phosphoric ester ester on on the the glycerol glycerol side side [36] [36] and phospholipase D D (PLD, (PLD, E. E.C.3.1.4.4)C.3.1.4.4) on on the the polar polar head head side side [37]. [37 ].Phospholipases Phospholipases are are widespread widespread in naturein nature playing playing very very different different physiological physiological roles roles [38,39]. [38,39 Enzymes]. Enzymes with with catalytic catalytic capacities, capacities, similar similar to cellto cell phospholipases phospholipases from from mammals mammals and andplants, plants, are also are alsofound found in bacteria in bacteria,, and t andhus, thus,that gives that giveseasy accesseasy access to specific to specific enzymes enzymes for for practical practical applications applications in in biocatalysis biocatalysis [40]. [40 ]. Detailed Detailed studies studies of of the structure, mechanism and and kinetics of phospholipases have been deeply reviewed [[4141–44]. All All the phospholipases withwith potentialpotential applicationsapplications are are hydrolytic hydrolytic enzymes enzymes that that are are often often secreted secreted outside outside the thecell. cell. Inprinciple, In principle, they they are ableare able to catalyze to catalyze both both the hydrolysisthe hydrolysis and theand reverse the reverse reaction reaction with with the ester the esterbond bond formation formation in the in presence the presence of the of suitable the suitable acceptor acceptor or donor. or donor.

Figure 2. EnzymesEnzymes acting acting on on phosphatidylcholine phosphatidylcholine (here (here reported reported as as 11-palmitoyl-2-linoleoyl--palmitoyl-2-linoleoyl- phosphatidylcholine) and their specific specific site of action.

ItIt should should be be noted noted that that in in recent recent years, there has been a partial shift in th thee topics of publications regardingregarding PLD activity activity;; in fact, more attention has been given to the production of of molecules with with a a specificspecific action rather than to the the general general screening screening of enzymatic enzymatic activities. In In this this review review,, the authors authors aimed to build an overview of the preparation of polar head modified PLs describing the main strategies to transform, by PLD-catalyzed transformations, the more abundant natural PL, PC, into these new PLs in order to extend the spectrum of PLs available for applications in basic research as well as in biomedical and biotechnological practice. Catalysts 2020, 10, x FOR PEER REVIEW 4 of 19 aimed to build an overview of the preparation of polar head modified PLs describing the main strategies to transform, by PLD-catalyzed transformations, the more abundant natural PL, PC, into theseCatalysts new2020 PLs, 10, 997in order to extend the spectrum of PLs available for applications in basic research4 of as 19 well as in biomedical and biotechnological practice.

2. Polar Polar Head Head Modificati Modificationon Polar head modified modified PLs (PX) find find applications in many many important important fields fields such such as as nutraceuticals, nutraceuticals, prodrugs, liposome technology and cosmetics. cosmetics. Phosphatidylation Phosphatidylation of of bi bioactiveoactive alcohols has been demonstrateddemonstrated,, inin fact, fact to, to be bea very a very suitable suitable strategy strategy in order in to order stabilize to stabilize and administer and administer active molecules active moleculesbecause the because phosphatidyl the phosphatidyl residue constitutes residue constitutes a nontoxic a nontoxic carrier moiety carrier with moiety a high with a affi highnity affinity for cell formembranes cell membranes and a great and hydrophobicitya great hydrophobicity which prevents which prevents bioactive bioactive moiety degradation.moiety degradation. PX canPX be can prepared be prepared starting starting from from PC PCvia via a PLD a PLD-catalyzed-catalyzed transphosphatidylation transphosphatidylation reaction. reaction. All All the bioconversions described in this review follow a quite simple and well well-established-established procedure which was already known in the middle of the 1990s1990s andand isis herehere presentedpresented inin SchemeScheme1 1[45 [45].].

Scheme 1. ReactionReaction scheme scheme of of a a phospholipase phospholipase D D ( (PLD)-mediatedPLD)-mediated polar polar head head transphosphatidylation transphosphatidylation of PC.

The reactions were performed on PC extracted from natural sources as a substrate. PC is one The reactions were performed on PC extracted from natural sources as a substrate. PC is one of of the main constituents of the so called lecithins provided commercially by the degumming step of the main constituents of the so called lecithins provided commercially by the degumming step of edible oils. Crude lecithins contain approximatively PLs and oil spanning in a range of 60 to 30 (w/w). edible oils. Crude lecithins contain approximatively PLs and oil spanning in a range of 60 to 30 (w/w). The principal components of PL fraction are PC, PE, PI and PA, which could be all separated and The principal components of PL fraction are PC, PE, PI and PA, which could be all separated and obtained with a high degree of purity by solvent partitioning and chromatographic steps [46]. obtained with a high degree of purity by solvent partitioning and chromatographic steps [46]. The enzymatic approach aiming to modify the polar head of PC, the most abundant natural The enzymatic approach aiming to modify the polar head of PC, the most abundant natural PL, PL, exploits PLD which is an extracellular hydrolytic enzyme with a broad substrate specificity. exploits PLD which is an extracellular hydrolytic enzyme with a broad substrate specificity. PLD PLD occurring in animals, plants and microorganisms catalyzes in vivo the hydrolysis of PC to choline occurring in animals, plants and microorganisms catalyzes in vivo the hydrolysis of PC to choline and phosphatidic acid (PA). However, some PLDs from particular sources possess a quite unique and phosphatidic acid (PA). However, some PLDs from particular sources possess a quite unique property among among hydrolytic hydrolytic enzymes enzymes which which is essential is essential in practical in practical applications applications such as the such preparation as the preparationof polar head-modified of polar head PLs-modified starting PLs from starting crude orfrom purified crude PC. or purified In fact, in PC. the In presence fact, in the of appropriatepresence of appropriatenucleophiles, preferably nucleophiles, primary preferably aliphatic alcohols, primary they aliphatic show a high alcohols, transphosphatidylation they show a capacity high transphosphatidylationeven in the presence of capacity water [33 even,45]. in When the presence the bioconversion of water [33,45]. is conducted When inthe the bioconversion presence of anis conductedappropriate in acceptorthe presence (an alcohol),of an appropriate transphosphatidylation acceptor (an alcohol), can occur transinphosphatidylation vitro with different can ratiosoccur inbetween vitro with PA different and the ratios new polar between head PA bearing and the PX,new depending polar head onbearing thenature PX, depending and concentration on the nature of andthe alcohol,concentration as well of as the on alcohol, the enzymatic as well as source on the and enzymatic on the overall source experimentaland on the overall conditions experimental [47,48]. conditionsIn the past [47,48]. 30 years, In a the great past number 30 years, of strainsa great ofnumber PLD have of strains been studied,of PLD have and thisbeen bioconversion studied, and this has bioconversionbeen exploited has in dibeenfferent exploited protocols in different to produce protocols a wide varietyto produce of new a wide products variety with of new very products different withapplications very different [49,50 ].applications However, an[49,50]. exact However, evaluation an of exact different evaluation PLDs isof di differefficultnt to PLDs illustrate is difficult because to illustratethe enzyme because source, the the enzyme alcohol source, used as the nucleophile, alcohol used the solventas nucleophile, system andthe thesolvent reaction system parameters and the provereaction to parameters be strongly interdependent.prove to be strongly Nevertheless, interdependent. the availability Nevertheless, of enzymes the availability with a wide of substrateenzymes withspecificity a wide and substrate high transfer specificity capacity and has high offered transfer very capacity convenient has access offered to naturalvery convenient and synthetic access polar to naturalhead modified and synthetic PLs. polar head modified PLs. The application of PLDs from bacterial sources has allowed the highly selective transfer of a nucleophilic component component (alcohol) (alcohol) in in a awater water medium medium to toPC PC with with the the formation formation of a of new a new PL and PL andPA inPA different in different ratios ratios [51]. [51 In]. In particular, particular, different different bacterial bacterial sources sources of of PLD PLD provided provided very very powerful enzymes with very different different transphosphatidylation potential.potential. In 1994,1994, D’Arrigo et al. identified identified three three StreptomyStreptomycesces species—PMF,species—PMF, PM43, PM43, PMR—as PMR—as microorganism microorganism producers producers of very of particularvery particular PLDs which PLDs werewhich applied were applied for the for transformation the transformation of the mostof the abundant most abundant natural natural PL, PC, PL, from PC, soyfrom beans soy beans into other into otherminor minor natural natural products products (PX), namely (PX), namely phosphatidylserine phosphatidylserine (PS), PE (PS), and phosphatidylglycerolPE and phosphatidylglycerol (PG) and also in a great number of unnatural compounds [52,53]. The reactions were usually carried out in water–organic solvent mixtures, and the extent of the competing hydrolytic reaction depended on the nature of the nucleophile and the reaction conditions. The presence of water in the reaction medium Catalysts 2020, 10, x FOR PEER REVIEW 5 of 19

(PG) and also in a great number of unnatural compounds [52,53]. The reactions were usually carried out in water–organic solvent mixtures, and the extent of the competing hydrolytic reaction depended Catalystson the nature2020, 10, of 997 the nucleophile and the reaction conditions. The presence of water in the reaction5 of 19 medium always caused the formation of the PA as a byproduct of the transphosphatidylation alwaysprocess, caused lowering the formation reaction yields of the andPA as forcing a byproduct the need of the to transphosphatidylation perform further purification process, steps. lowering The reactionpurification yields of and this forcing class the of need compounds to perform was further not purification straightforward steps. and The requiredpurification different of this classchromatographic of compounds and was selective not straightforward precipitation and steps. required Thus, di fromfferent a chromatographic synthetic point ofand view, selective the precipitationpossibility to steps.enhance Thus, the fromselectivity a synthetic of the pointreaction of view,could the allow possibility to simplify to enhance the preparation the selectivity of these of thecompounds reaction on could a relatively allow to larger simplify scale the leading preparation to lower of theseproduction compounds costs. on a relatively larger scale leading to lower production costs. 2.1. Natural PLs 2.1. Natural PLs 2.1.1. Phosphatidylserine 2.1.1. Phosphatidylserine Phosphatidylserine (PS, PX-1) has achieved a great commercial interest for a long time as it is a membranePhosphatidylserine PL ubiquitous (PS,ly presentPX-1) hasin all achieved cellular amembranes, great commercial especially interest in the for brain a long, and time is involved as it is a membranein many neurological PL ubiquitously processes present [30]. in all It is cellular a molecule membranes, that presents especially many in the nutritional brain, and and is involved medical infunctions many neurologicalas low levels processesof PS is linked [30]. to It memory is a molecule problems, that to presents Alzheimer many’s and nutritional other mental and diseases. medical functionsIt is demonstrated as low levels that ofthe PS assumption is linked to of memory PS leads problems, to positive to benefits Alzheimer’s in terms and otherof mind mental and memory diseases. Itenhancement. is demonstrated It reduces that the the assumption age-related of PS decline leads in to positivemental function, benefits in it terms prevents of mind Alzheimer and memory’s and enhancement.dementia, it is considered It reduces thea mood age-related enhancer decline to cure in depression, mental function, it improves it prevents athletic Alzheimer’s performance, and it dementia,is a stress reducer it is considered and it is a an mood important enhancer ingredient to cure depression,of liposomes it improvesused in drug athletic delivery performance, [54]. The itmain is a stressdrawback reducer is linked and itto is its an low important availability ingredient in nature of and liposomes high costs used of in extraction drug delivery from nat [54ural]. The sources main drawback[42]. is linked to its low availability in nature and high costs of extraction from natural sources [42]. The most commoncommon methodmethod describeddescribed by by di differentfferent authors authors in in the the 1990s 1990s was was the the synthesis synthesis of PSof PS by usingby using diff erent different PLD PLD solutions solutions (from (fromStreptomyces StreptomycesPMF, cabbage,PMF, cabbage,Streptomyces Streptomyces prunicolor prunicolor) in biphasic) in reactionbiphasic mixturesreaction (asmixtures shown (as in Schemeshown 2in)[ Scheme33,55]. The2) [3 best3,55]. results The werebest results obtained were with obtained the PLD with solution the fromPLD solutionStreptomyces fromsp. Streptomyces In detail, a sp. mixture In detail, of enzymatic a mixture solution of enzymatic with 0.2 solution M phosphate with 0.2 bu Mffer phosphate at pH 5.6 containingbuffer at pH 3.4 5.6 M containing L-serine and 3.4 M CaCl L-serine2 was and added CaCl to2 anwas initial added solution to an initial of PC solution 17.8 M inof ethylPC 17.8 acetate. M in Theethyl main acetate. drawback The main of the drawback traditional of the systems traditional was, assystems usual, thewas concomitant, as usual, the formation concomitant of PA formation with the desiredof PA with PS andthe desired the necessary PS and purification the necessary steps. purification steps.

Scheme 2. Schematic equation of the synthesis of phosphatidyserine (PX-1PX-1).

The modificationmodification and the improvements of the reaction conditions for the synthesis of PS have been the the object object of of many many studies studies taking taking advantage advantage of new of strategies new strategies in the biocatalytic in the biocatalytic field. In particular, field. In theparticular, screening the of screening the use ofof a the great use number of a great of novel number eco-friendly of novel solventseco-friendly such solvents as ionic such liquids as (ILs);ionic deepliquids eutectic (ILs); deep solvents eutectic (DES); solvents biobased (DES) solvents; biobased such assolventsΥ-valerolactone, such as Υ-valerolactone, limonene and p-cymenelimonene wasand performed.p-cymene was On performed. this topic, in On 2012, this D’Arrigotopic, in 2012 et al., D’Arrigo started to et investigate al. started forto investigate the first time for the the possibility first time tothe switch possibility PLD-catalyzed to switch reactions PLD-catalyzed from water reactions to a nonaqueous from water green to a systemnonaqueous [56]. Their green work system aimed [56]. to use the IL [BMIM] [PF ] as an alternative solvent to perform the PLD-mediated enzymatic conversion in Their work aimed to use6 the IL [BMIM] [PF6] as an alternative solvent to perform the PLD-mediated orderenzymatic to obtain conversion the modification in order of tothe obtain enzyme the selectivity modific withation the of suppression the enzyme of unwanted selectivity hydrolytic with the sidesuppression reactions, of leadingunwanted to anhydrolytic easier product side reactions, recovery leading by a simple to an solventeasier product extraction. recovery The reduction by a simple or absencesolvent extraction. in water leads, The inreduction fact, to the or absenceabsence of in the water competitive leads, inhydrolysis fact, to the reaction absence making of the competitive the product purificationhydrolysis reaction much easier making and the cheaper product due purification to the limited much use of easier organic and solvents, cheaper because due to the purificationlimited use stepsof organic could be solvents performed, because without the very purification long chromatographic steps could separations. be performed The without bioconversion very long was performedchromatographic by dissolving, separations. at first, The PC bioconversion in toluene at was 40 ◦ performedC. A mixture by ofdissolving IL/0.1 M,sodium at first, PC acetate in toluene buffer (90/10) at pH 5.6 containing 3 M of L-serine and CaCl2 0.1 M was added to the solution of PC under magnetic stirring. PLD from Streptomyces PMF was then added, and the bioconversion was stirred for Catalysts 2020, 10, 997 6 of 19

24 h at 40 ◦C, yielding 91.4% of PS with a complete suppression of unwanted hydrolytic side reaction, which resulted in an easier final product purification. In the same period, Duan et al. developed the reactions in two other reaction systems to prepare PS without the formation of PA with an overall yield over 90% using Υ-valerolactone in 2012 [57] and 2-methyltetrahydrofuran in 2013 [58]. The undesired hydrolytic side reaction problem was also addressed by Chen et al. in 2013, who developed the conditions to perform the PLD transphosphatidylation of PC in a microaqueous water-immiscible organic solvent in order to prevent undesired hydrolysis of PLs [59]. The bioconversion was conducted with PLD from Streptomyces racemochromogenes at 30 ◦C for 8 h, with an L-serine to PL ratio of 20:1, the solvent system contained 6% of water in butyl acetate. This bioconversion yielded 88% of the desired product reaching a moderately complete suppression of the undesirable hydrolysis. More recently, Yang et al. [60] described the use of deep eutectic solvents (DES) as an alternative inexpensive, environmentally friendly, enzyme compatible solvent to conduct the PS synthesis. Different types of DES were tested, as a general procedure, the bioconversion was carried out in a batch reactor at 40 ◦C, the reaction mixture was composed of PC, DES, L-serine, PLD and water (0.5%). The reaction was conducted using choline chloride/ethylene glycol as DES gave the best results yielding PS over 90%. Very recently, Qin et al. performed the synthesis of PS in the eco-friendly and green solvent, cyclopentyl methyl ether, using a recombinant PLD from Streptomyces chromofuscus expressed in Escherichia coli which allowed a yield of 84.4% of conversion of PC in a scale-up production of 1 L volume to be reached [61].

2.1.2. Phosphatidylglycerol and Cardiolipin Phosphatidylglycerol (PG, PX-2) is one of the most abundant PLs found in natural membranes. It possesses an excellent liposome forming ability and has been studied from a long time—since 1985—as an emulsifier for drug delivery systems [62–64]. In fact, PG is known to have unique surfactant/lubricant properties, particularly when mixed with appropriate proteins. It has been found, for example, in relatively large amounts in mammalian lungs compared to other mammalian membranes pushing the research on the development of surfactant preparations for therapeutic practices for the relief of a variety of diseases such as neonatal respiratory distress syndrome [65]. Although PG is a naturally occurring PL, it is found only in a small amount in nature, and its enzymatic preparation has been performed by different groups [66]. For example, Piazza et al. demonstrated that with PLD from Streptomyces sp., PG was obtained in a nearly quantitative yield when the molar ratio of glycerol to PC was at least 5 to 3 showing that this PLD had a higher selectivity for glycerol than in cabbage PLD [67]. Moreover, during the preparation of PG catalyzed by PLD from Streptomyces sp. (as shown in Scheme3), D’Arrigo et al., in 1996 [ 68], observed that the prepared PG under appropriate conditions could be transformed into diphosphatidyglycerol (cardiolipin, CLP, PX-3)[69]. This molecule is an acidic lipoprotein that is abundant in the inner mitochondrial membrane and is required for normal respiratory chain enzymic activity [70]. It is particularly abundant in the mammalian heart. It is also known to be the essential lipid of the antigen in the serodiagnosis of syphilis. The synthesis was performed starting from PC dissolved in dichloromethane, which was treated with PLD in sodium acetate buffer 0.1 M at pH 5.6 with CaCl2 0.1 M containing an excess of glycerol (8 M). The bioconversion was left stirring at 200 rpm at 25 ◦C for 7 h, yielding PG in high yields. The obtained PG was dissolved in CH2Cl2 and treated again with fresh PLD in the same buffer. This mixture was left stirring for 24 h at 37 ◦C. Then, the biphasic mixture was allowed to separate in order to withdraw the aqueous phase and replaced with a fresh enzymatic solution with a complete conversion in PX-3 after 5 days. Catalysts 2020, 10, 997 7 of 19 Catalysts 2020, 10, x FOR PEER REVIEW 7 of 19 Catalysts 2020, 10, x FOR PEER REVIEW 7 of 19

Scheme 3. Schematic equation of the synthesis of phosphatidylglycerol (PX-2) and cardiolipin (PX-3). SchemeScheme 3. 3.Schematic Schematic equation equation of of thethe synthesissynthesis of phosphatidylglycerol (PX (PX-2-2) )and and cardiolipin cardiolipin (PX (PX-3-3). ).

2.1.3.2.1.3. PhosphatidylethanolaminePhosphatidylethanolamine PhosphatidylethanolaminePhosphatidylethanolamine (PE, (PE, PXPX-4PX--44)) is is another another majormajor PLPL inin thethe membranesmembranes ofof of eukaryoticeukaryotic eukaryotic cells.cells. cells. TheThe importance importance importance of of of PE PE PE metabolism metabolism metabolism in mammalian in in mammalian mammalian health health healthhas recently has has recently emergedrecently followingemerged emerged itsfollowing following association its its associationwithassociation Alzheimer’s, with with Alzheimer Alzheimer Parkinson’s’s’s,, and Parkinson Parkinson nonalcoholic’s’s andand liver nonalcoholic nonalcoholic diseases, as liver liver well diseases, diseases,as with the asas virulence well well as as of with with certain the the virulencepathogenicvirulence ofof organisms certaincertain pathogenicpathogenic [71,72]. Moreover, organismsorganisms PE [71,72].[71,72]. has a Moreover, significantMoreover, PE influencePE hashas aa significantsignificant on the heart influenceinfluence to prevent onon cellthethe heartdamage.heart to to prevent preventThis product cell cell damage. damage. also presents This This good product product antioxidant also also presents presents activity good good against antioxidant antioxidant free radicals, activity activity which against against is another free free radicalscharacteristicradicals,, whichwhich which isis anotheranother could characteristiccharacteristic provide beneficial whichwhich e ff couldcouldects to provideprovide human beneficialbeneficial health. effectseffects toto humanhuman health.health. TheThe productionproduction ofof of PEPE PE fromfrom from naturalnatural natural sourcessources sources byby by solventsolvent solvent extractionextraction extraction constitutesconstitutes constitutes thethe the mainmain main strategystrategy strategy toto recovertorecover recover it it even iteven even if if if the the the purity purity purity of of of PE PE PE does does does not not not always always fit fitfit industrial industrial requirements. requirements. requirements. Chromatographic Chromatographic methomethodsmethodsds allowedallowed thethe the obtainmentobtainment obtainment ofof of moremore more purifiedpurified purified PEPE PE samplessamples samples eveneven even ifif iftheythey they requiredrequired required highhigh high volumesvolumes volumes ofof organicoforganic organic solvents solvents solvents and and and multistep multistep multistep procedures procedures procedures [73]. [73]. [73]. PLD PLD PLD-catalyzed--catalyzedcatalyzed transformations transformations transformations of of of natural natural PC PC representsrepresents thethe bestbest wayway toto prepareprepare PEPE ininin highhighhigh puritypuritypurity (see(see(see SchemeSchemeScheme4 4).4).).

Scheme 4. Schematic equation of the synthesis of phosphatidylethanolamine (PX-4PX-4,, PE).PE). Scheme 4. Schematic equation of the synthesis of phosphatidylethanolamine (PX-4, PE). The first results were published by Juneja et al. in 1988; they conducted the transphosphatidylation TheThe first first results results were were published published by by Juneja Juneja et et al. al. in in 1988 1988;; theythey conducted conducted the the oftransphosphatidylation PC to PE by testing seven of PC di tofferent PE by sources testing of seven phospholipase different sources D, one ofof which phospholipase was obtained D, one from of transphosphatidylationcabbage, whilst the others of PC were to PE all by obtained testing fromsevenStreptomyces different sourcessp. All of thephospholipase bioconversions D, one were of whichwhich waswas obtainedobtained fromfrom cabbagecabbage,, whilstwhilst thethe othersothers werewere allall obtainedobtained fromfrom StreptomycesStreptomyces sp.sp. AllAll thethe performedbioconversions at 30 were◦C for performed 1 h in a biphasic at 30 °C system for 1 h composedin a biphasic of ethylsystem acetate composed and a of bu ethylffer containing acetate and the a bioconversionsenzyme. Different were parameters performed such at 30 as °C the for type 1 h of in bu aff biphasicer (concentration system composed and pH), andof ethyl the concentrationacetate and a bufferbuffer containingcontaining thethe enzyme.enzyme. DifferentDifferent parametersparameters suchsuch asas thethe typetype ofof bufferbuffer (concentration(concentration andand pH),pH), of PC, ethanolamine and CaCl2, were tested. The best observed conditions in terms of reaction and the concentration of PC, ethanolamine and CaCl2, were tested. The best observed conditions in andselectivity the concentration around 100% of were PC, obtained,ethanolamine on one and hand, CaCl with2, were PLD tested. from Streptomyces The best observedsp. and ethanolamineconditions in termsterms ofof reactionreaction selectivityselectivity aroundaround 100%100% werewere obtainedobtained,, onon oneone handhand,, withwith PLDPLD fromfrom StreptomycesStreptomyces 4.9sp and M, and ethanolamine on the other 4.9 hand, M, and with on PLD the other from cabbagehand, with with PLD ethanolamine from cabbage 2 M with [74]. ethanolamine 2 M sp andD’Arrigo ethanolamine et al. prepared 4.9 M, and PE on from the PC other in a hand biphasic, with system PLD from (ethyl cabbage acetate/ NaAcOwith ethanolamine buffer 0.1 M 2 pH M [74].[74]. 5.6, 0.1 M CaCl2, 1 M ethanolamine) with PLD from Streptomyces PMF and PM43 with a very high D’ArrigoD’Arrigo etet al.al. preparedprepared PEPE fromfrom PCPC inin aa biphasicbiphasic systemsystem (ethyl(ethyl acetate/NaAcOacetate/NaAcO bufferbuffer 0.10.1 MM pHpH selectivity (PE/PA = 23 and 22, respectively) and yield (96% and 92%, respectively) at 37 ◦C in 3 h [53]. 5.6, 0.1 M CaCl2, 1 M ethanolamine) with PLD from Streptomyces PMF and PM43 with a very high 5.6, 0.1Dippe M CaCl et al.2, performed 1 M ethanolamine the enzymatic) with transphospatidylation PLD from Streptomyces of 1,2-dioleoyl-sn-glycerophosphocholine PMF and PM43 with a very high selectivityselectivity (PE/PA(PE/PA == 2323 andand 2222,, resrespectively)pectively) andand yieldyield (96(96%% andand 9292%%,, respectively)respectively) atat 3737 °C°C inin 33 hh [53].[53]. with N-Dippe or C2-substituted et al. perfo derivativesrmed of the ethanolamine enzymatic (diethanolamine, transphospatidylation triethanolamine, of 1,2- serinol,dioleoyl Tris,-sn- Bistris),Dippe by testing et al. two perfodifferentrmed PLDs the from enzymaticStreptomyces transphospatidylationsp. and cabbage, aiming of to produce 1,2-dioleoyl a series-sn- glycerophosphglycerophosphocholineocholine with with N N-- oror C2 C2--substitutedsubstituted derivatives derivatives of of ethanolamine ethanolamine (diethanolamine, (diethanolamine, oftriethanolamine, ethanolamine derivatives serinol, Tris, with Bistris), identical by testing charge two but with different diff erent PLDs volumes from Streptomyces with an interest sp. and in triethanolamine,biotechnological practices serinol, [ Tris,75]. The Bistris), production by testing of these two new different PLs was PLDs performed from inStreptomyces a two phase sp. system and cabbage,cabbage, aiming aiming to to produce produce a a series series of of ethanolamine ethanolamine derivatives derivatives with with identical identical chargecharge but but with with consistingdifferent volume of NaOAcs with bu anffer interest 300 mM in at biotechnological pH 5.6 containing practices 120 mM [75]. of CaClThe 2productionand 2.8 mM of ofthese the new acceptor PLs differentalcohol and volume diethyls with ether an tointerest which in the biotechnological enzyme was added. practices These [75]. bioconversions The production were of these conducted new PLs at waswas performedperformed inin aa twotwo phasephase systemsystem consistingconsisting ofof NaOAcNaOAc bufferbuffer 300300 mMmM atat pHpH 5.65.6 containingcontaining 120120 mM of CaCl2 and 2.8 mM of the acceptor alcohol and diethyl ether to which the enzyme was added. mM of CaCl2 and 2.8 mM of the acceptor alcohol and diethyl ether to which the enzyme was added.

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30 ◦C, shaken at 400 rpm for 5 h in the case of the PLD from Streptomyces sp. and 8 h in the case of PLD Thesefrom cabbage. bioconversions were conducted at 30 °C, shaken at 400 rpm for 5 h in the case of the PLD from Streptomyces sp. and 8 h in the case of PLD from cabbage. 2.2. Synthetic PLs 2.2. Synthetic PLs The synthetic PLs, which will be described in this section, are summarized in Table1. The PLD sourceThe and synthetic the reaction PLs, which time as will well be as described its main applicationin this section, is also are reported summarized in Table in Table1. 1. The PLD source and the reaction time as well as its main application is also reported in Table 1. Table 1. Synthetic phospholipids (PLs) with essential preparation parameters (PLD source, incubation Tabletime, yield) 1. Synthetic and their phospholipids main applications. (PLs) with essential preparation parameters (PLD source, incubation time, yield) and their main applications. Compound PLD Source Incubation Time (h) Yield (%) Application CompoundPX-5 PLD SourceStreptomyces lydicusIncubation Time (h)24 Yield (%) 90Application Antioxidant PX-5 PX-6 StreptomycesStreptomyces lydicus lydicus 24 290 94Antioxidant Antioxidant PX-6 PX-7 Streptomyces Streptomyceslydicus sp.2 494 18Antioxidant Tyrosinase inhibitor PX-7 PX-8 StreptomycesStreptomyces sp. sp.4 318 60Tyrosinase Tyrosinase inhibitor inhibitor PX-8 PX-9 StreptomycesStreptomyces sp. sp.3 360 61Tyrosinase Cytotoxic inhibitor compound PX-9 PX-10 StreptomycesStreptomyces sp. sp.3 2461 90Cytotoxic Antibacterialcompound PX-10 PX-11 StreptomycesStreptomyces sp. sp.24 2490 73Antibacterial Antibacterial PX-11 PX-12 StreptomycesStreptomyces sp. sp.24 2473 54Antibacterial Antibacterial PX-12 PX-13 StreptomycesStreptomyces sp. sp.24 2454 17Antibacterial Antibacterial PX-13 PX-14 StreptomycesActinamadure sp. sp.24 2417 97Antibacterial Intracellular antioxidant PX-14 PX-15 ActinamadureStreptomyces sp. sp.24 297 95Intracellular Preparation antioxidant of anionic liposomes PX-15 PX-16 StreptomycesStreptomyces sp. sp.2 295 67Preparation Preparation of anionic of anionic liposomes liposomes PX-16 PX-17 StreptomycesStreptomyces sp. sp.2 267 23Preparation Preparation of anionic of anionic liposomes liposomes PX-17 Streptomyces sp. 2 23 PreparationImportant of anionic compound liposomes in di fferent PX-18 Streptomyces sp. 22 12.5 PX-18 Streptomyces sp. 22 12.5 Important compound inhuman different organs human organs PX-20 PX-20 ActinamadureActinamadure sp. sp.48 4887 87Preparation Preparation of new of liposomes new liposomes PX-21 PX-21 StreptomycesStreptomyces sp. sp.12 1294 94Anesthetic Anesthetic and sedative and sedative PX-22 PX-22 StreptomycesStreptomyces sp. sp.24 2496 96Anti-inflammatory Anti-inflammatory

2.2.12.2.1.. 6 6-Phosphatidyl--Phosphatidyl-Ll--AscorbicAscorbic Acid Nagao et et al. al. worked worked,, in early in early times times (1991) (1991),, on the on cellular the cellular defens defensee system systemagainst againstoxygen toxicity oxygen andtoxicity especially and especially against membrane against membrane lipid peroxidation lipid peroxidation [76]. The [76 membrane]. The membrane defense defense against oxygenagainst radicals,oxygen radicals, which occurs which occursat the atinterface the interface between between the aqueous the aqueous phase phase and and the the membrane membrane lipids, lipids, is considerablyis considerably important. important. The The idea idea of ofthe the authors authors was was to to prepare prepare a anew new PL PL with with antioxidant antioxidant activity activity bearing an appropriate antioxidant antioxidant residue residue as as a a polar polar head head;; 6 6-phosphatidyl--phosphatidyl- Ll--ascorbicascorbic acid acid ( (PPX-5X-5)) was selected because LL-ascorbic-ascorbic acid is among the important antioxidants in the cellular defendefensese against oxygen radicals, as well presenting a primary hydroxyl group available for PLD catalysed transphosphatidylation. The The bioconversion bioconversion was was performed performed i inn a a biphasic biphasic system, system, where where diethyl ether was selectedselected toto bebe the the best best organic organic solvent solvent as as it reducedit reduced the the collateral collateral production production of phosphatidic of phosphatidic acid acid(as shown (as shown Scheme Scheme5). The 5). reaction The reaction mixture mixture contained contained a solution a solution of PC from of PC egg from yolk egg 10 yolk mM in10 diethylmM in diethylether, 2 ether, M L-ascorbic 2 M L-ascorbic acid adjusted acid adjusted to pH 4.5 to withpH 4.5 KOH with and KOH a PLD and solutiona PLD solution from Streptomyces from Streptomyces lydicus lydicusin sodium in sodium acetate buacetateffer 10 buffer mM, pH10 mM, 5.1. The pH mixture5.1. The was mixture left shaking was left at shaking 37 ◦C for at 24 37 h °C to obtainfor 24 ah PCto obtainconversion a PC ofconversion 90%. of 90%.

l SchemeScheme 5. 5. SchematicSchematic equation equation of of the the synthesis synthesis of of 6 6-Phosphatidyl--Phosphatidyl- L-Ascorbic-Ascorbic Acid (PX-5PX-5). 2.2.2. Cyclic Hydroxylated Polar Head Phospholipids 2.2.2. Cyclic Hydroxylated Polar Head Phospholipids For many years, the use of PLD for the preparation of new synthetic PLs has been a widely studied For many years, the use of PLD for the preparation of new synthetic PLs has been a widely topic. However, most of the literature found before 2010 reports the screening and optimization of the studied topic. However, most of the literature found before 2010 reports the screening and optimization of the PLD activity on a great number of potential substrates without evaluating the real applications of the obtained product. The unique exceptions were the molecules reported in

Catalysts 2020, 10, 997 9 of 19

Catalysts 2020, 10, x FOR PEER REVIEW 9 of 19 PLD activity on a great number of potential substrates without evaluating the real applications of the obtainedScheme 6 product., which bore The uniquea cyclic exceptionshydroxylated were polar the molecules head and reportedwere reported in Scheme in 19946, which by the bore groups a cyclic of hydroxylatedKoga and Takami polar for head specific and were uses. reported in 1994 by the groups of Koga and Takami for specific uses.

Scheme 6. SyntheticSynthetic PLs ( PXPX-6-6/9/9)) presenting a targeted application.

Compound PX-6PX-6 (1,2-diacyl-(1,2-diacylsn-sn-glycero-3-phospho-2-glycero-3-phospho-02′-hydroxyethyl-2-hydroxyethyl-02,5′,5′,7′,8′0,70,80-tetramethyl-6-tetramethyl-6′0-- hydroxychroman, namednamed also also phosphatidylchromanol) phosphatidylchromanol was) was synthesized synthesized in 1994 in 1994 by Koga by Koga et al. byet al using. by PLDusing from PLDStreptomyces from Streptomyces lydicus ,lydicus and the, and product the product obtained obtained presented presented a very gooda very antioxidant good antioxidant activity againstactivity lipid against peroxidation lipid peroxidation in PL membranes in PL membranes [77]. PX-6 [77].was preparedPX-6 was from prepared egg yolk from PC egg dissolved yolk PC in diethyldissolved ether in with diethyl 2,5,7,8-tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman; ether with 2,5,7,8-tetramethyl-6-hydroxy-2-(hydroxyethyl)chroman this solution was added; this to PLDsolution previously was added dissolved to PLDin previously 10 mM acetate dissolved buff erin at10 pHmM 5.1 acetate with 10buffer mM at CaCl pH 25.1. The with incubation 10 mM CaCl was2. performedThe incubation at 37 was◦C for performed 2 h, and theat 37 desired °C for product 2 h, and was the obtained desired withproduct a 94% was yield. obtained with a 94% yield.Compounds PX-7 (1,2-dipalmitoyl-3-sn-phosphatidylarbutin) and PX-8 (1,2-dipalmitoyl-3- sn-phosphatidylkojicCompounds PX-7 acid) (1,2-dipalmitoyl were produced-3-sn-phosphatidylarbutin) in the same year by and Takami PX-8 et (1,2 al.-dipalmitoyl by using-3 PLD-sn- fromphosphatidylkojicStreptomyces acid)sp. Thesewere produced compounds in were the same demonstrated year by Takami to be tyrosinase et al. by usinginhibitors PLD in from the sameStreptomyces manner sp as. These the polarcompounds heads arbutinwere demonstrated and kojic acid, to be which tyrosinase unfortunately inhibitors werein the water-solublesame manner andas the instable polar heads for cosmetic arbutin use.and kojic Their acid, derivatization which unfortunately in PX-7 and werePX-8, waterrespectively,-soluble and allowed instable their for actioncosmetic in use cosmetics. Their derivatization and pharmaceuticals in PX-7 and to PX be-8 tested, respectively in order, allowed to prevent their overproductionaction in cosmetics of melaninand pharmaceuticals in epidermal to cells be tested by inhibiting in order tyrosinaseto prevent overproduction activity [78]. PX-7 of melaninwas prepared in epidermal by mixing cells 1,2-Dipalmitoyl-by inhibiting tyrosinasesn-glycero-3-phosphocholine activity [78]. PX-7 was (DPPC) prepared with arbutin by mixing in a 1,2 biphasic-Dipalmitoyl system-sn composed-glycero- of3- ethylphosphocholine acetate and (DPPC) sodium acetatewith arbutin buffer 0.2in a M biphasic at pH 5.6 system with 4% composed of CaCl2 containingof ethyl acetate PLD. Afterand sodium having stirredacetate thebuffer reaction 0.2 M atat 35pH◦ C5.6 for with 4 h, 4% the of desiredCaCl2 containing product was PLD. obtained After having with anstirred 18% the yield, reaction and the at undesired35 °C for 4 PAh, the was desired obtained product with awas 48% obtained yield. In with the casean 18 of% productyield, andPX-8 the, theunde reactionsired PA conditions was obtained were slightlywith a 48 modified;% yield. theIn the initial case DPPC of product was mixed PX-8, with the reaction kojic acid conditions and still treated were slightly with PLD, modified but in; thisthe case,initial the DPPC biphasic was mixture mixed with was kojic composed acid and of diethyl still treated ether and with sodium PLD, but acetate in this bu ff caseer 0.2, the M biphasic without CaClmixture2. The was reaction composed was of performed diethyl ether for and 3 h, sodium and the acetate desired buffer product 0.2 wasM without obtained CaCl with2. The a 60% reaction yield withwas performed a much lower for production3 h, and the of desired PA (9%). product was obtained with a 60% yield with a much lower productionCompound of PA PX-9(9%)., phosphatidylgenipin, still studied by Takami et al. by using PLD from StreptomycesCompounsp.d presentedPX-9, phosphatidylgenipin, a very high cytotoxic still action studied against by Takami Hela cells, et al. human by using embryonic PLD from lung fibroblastStreptomyces and sp MT-4. presented cells [79 ].a very The insertion high cytotoxic of genipin action into against the phosphatidyl Hela cells, backbone human deeplyembryonic enhanced lung itsfibroblast cytotoxicity and towards MT-4 cells the three [79]. cellThe lines insertion because of it genipin represented into athe very phosphatidyl good carrier ba intockbone cells. Fordeeply the preparationenhanced its of cytotoxicityPX-9, DPPC towards was incubated the three with cell genipin lines because in a biphasic it represented mixture composed a very good of ethyl carrier acetate into andcells. sodium For the acetatepreparation buffer of 0.2PX- mM9, DPPC at pH was 5.4 incubated with 4% ofwith CaCl genipin2 and in PLD. a biphasic After 3 mixture h of incubation composed at 35of ethyl◦C under acetate stirring, and sodium the product acetate was buff obtaineder 0.2 mM with at a pH yield 5.4 of with 61%. 4% of CaCl2 and PLD. After 3 h of incubation at 35 °C under stirring, the product was obtained with a yield of 61%. 2.2.3. Phosphatidylated Terpenes 2.2.3.In Phosphatidylated 2008, Yamamoto Terpenes et al. investigated the transphosphatidylation of PC with terpenes in order to introduceIn 2008, functionalYamamoto terpenes et al. investigated into PLs for the food transphosphatidylation and cosmetic applications of PC (as with shown terpen ines Scheme in order7). Theyto introduce prepared functional new phosphatidylated terpenes into PLs monoterpenes for food and cosmetic containing applications geraniol ( PX-10(as shown), geranylgeraniol in Scheme 7). (TheyPX-11 prepared), phytol new (PX-12 phosphatidylated) and farnesol (PX-13 monoterpenes) which were containing known togeraniol show a(PX markedly-10), geranylgeraniol antibacterial (PX-11), phytol (PX-12) and farnesol (PX-13) which were known to show a markedly antibacterial activity and an antiproliferative effect on human cancer cells [80]. The aim was to exploit these peculiar characteristics and use them for industrial applications in the food and cosmetic industries.

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Catalysts 2020, 10, x FOR PEER REVIEW 10 of 19 Catalysts 2020, 10, x FOR PEER REVIEW 10 of 19 activity and an antiproliferative effect on human cancer cells [80]. The aim was to exploit these peculiarDifferent characteristics bioconversion and conditions use them were for industrial studied, the applications reactions in were the food carried and out cosmetic both in industries. biphasic Disystemsfferent containing bioconversion ethyl conditions acetate/water were studied,or directly the in reactions water without were carried organic out solvents. both in biphasic Different systems PLDs containingwere tested, ethyl deriving acetate / fromwater Streptomyces or directly in spwater., Streptomyces without organic chromofusc solvents.us, c Diabbagefferent PLDsand peanut. were tested, The derivingbioconversions from Streptomyces were conductedsp., Streptomyces, at first, by dissolving chromofuscus PC, cabbageand the terpene and peanut. in ethyl The acetate bioconversions followed by the addition of PLD in buffer containing CaCl2 and albumin. For the PLDs deriving from cabbage wereby the conducted, addition of at PLD first, in by buffer dissolving containing PC and CaCl the2 and terpene albumin. in ethyl For acetatethe PLDs followed deriving by from the additioncabbage and peanut, the buffer was sodium acetate 0.2 M at pH 5.6; for the other two PLDs, the buffer was ofand PLD peanut in bu, theffer buffer containing was sod CaClium2 and acetate albumin. 0.2 M Forat pH the 5.6 PLDs; for derivingthe other fromtwo PLDs cabbage, the and buffer peanut, was theTris bu-HClffer at was pH sodium8. The reactions acetate 0.2were M conducted at pH 5.6; forat 37 the °C, other 350– two400 rpm PLDs, in the budarkffer for was 24 Tris-HClh. Only the at PLD from Streptomyces sp. revealed to be a suitable enzyme for terpene-PL synthesis, the yield of pHPLD 8. from The reactionsStreptomyces were sp. conducted revealed to at 37be ◦aC, suitable 350–400 enzyme rpm in for the terpene dark for-PL 24 synthesis h. Only the, the PLD yield from of Streptomyceswhich decreasedsp. revealed with the to chain be a suitable length of enzyme the terpene for terpene-PL used going synthesis, from 90 the% for yield PX of-10 which to 17 decreased% for PX- with13. the chain length of the terpene used going from 90% for PX-10 to 17% for PX-13.

Scheme 7. Schematic equation of the terpenes terpenes—phosphatidylderivatives—phosphatidylderivatives (PX-10PX-10/13/13).).

2.2.4. Phosphatidyltyrosol More recently recently,, in 2013, 2013, with with the the same same goal goal of of the the previous previous work work reported reported in inSection Section 2.2.3, 2.2.3 of, ofimproving improving the the intracellular intracellular antioxidant antioxidant defen defensese systems, systems, Victor Victor Casado Casado et et al. al. decided decided to introduce the tyrosoltyrosol residue residue in in the the PLs PLs polar polar head; head in; fact,in fact tyrosol, tyrosol was was known known to exert to powerfulexert powerful protective protective effects againsteffects against oxidative oxidative injuries injuries in cell systemsin cell systems [81,82]. [81,82]. Authors Authors pointed pointed out that out the that effectiveness the effectiveness of tyrosol of intyrosol some in biological some biological systems was systems related was to related its capability to its capabilityto penetrate to the penetrate cells. Then, the cells. they studied Then, they the productionstudied the andproduction scale-up and of phosphatidyltyrosol scale-up of phosphatidyltyrosol (PX-14) aiming (PX to-14 exploit) aiming the to known exploit antioxidative the known eantioxidativeffect of tyrosol, effect in particular, of tyrosol, by in augmenting particular, by its augmenting effectiveness its through effectiveness lipophilization through which lipophilization enhanced itswhich solubility enhanced and its capability solubility of and penetrating capability cells. of penetrating cells. This bioconversion, reported in Scheme 8 8,, was was performed performed using using a a food food grade grade PLD PLD from from Actinamadure sp.sp. inin aa GRASGRAS (Generally(Generally Recognized Recognized as as Safe) Safe) biphasic biphasic reaction reaction medium medium composed composed of anof aqueousan aqueous phase phase and and ethyl ethyl butyrate butyrate (1:2 (1:2v/ vv/v).). The The aqueous aqueous phase phase consisted consisted of of a a sodiumsodium acetateacetate buffer buffer 0.2 M pHpH 5.6,5.6, 208208 mMmM ofof tyrosol,tyrosol, 6767 mMmM CaClCaCl2,, andand thethe bioconversionbioconversion waswas conductedconducted at 4040 °CC and 0.2 M pH 5.6, 208 mM of tyrosol, 67 mM CaCl22, and the bioconversion was conducted at 40 ◦°C and 200 rpm. rpm. The The studied studied process process was was also alsoscaled-up scaled in-up a 1 Lin thermostated a 1 L thermostated stainless steel stainless reactor steel containing reactor 660containing g of reaction 660 g mixture of reaction under mixture N to avoidunder oxidation, N2 to avoid allowing oxidation, the obtainment allowing the of theobtainment highly purified of the containing 660 g of reaction mixture2 under N2 to avoid oxidation, allowing the obtainment of the desiredhighly purified product desiredPX-14 with product a 97% PX yield-14 with in mass. a 97% yield in mass.

Scheme 8. Schematic equation of the synthesis of phosphatidyltyrosol (PX-14). Scheme 8. Schematic equation of the synthesis of phosphatidyltyrosol (PX-14).

2.2.5. Phosphatidyl Saccharides Another interesting application of new PLs was studied by Song et al. in 2013; they prepared phosphatidyl saccharides (PX-15 to PX-17)—reported in Scheme 9—for the preparation of anionic

Catalysts 2020, 10, 997 11 of 19

2.2.5. Phosphatidyl Saccharides Catalysts 2020, 10, x FOR PEER REVIEW 11 of 19 Another interesting application of new PLs was studied by Song et al. in 2013; they prepared phosphatidylnanoliposomes saccharides with enhanced (PX-15 stabilityto PX-17 [83].)—reported They studied in Scheme the correlation9—for the preparationbetween enzymatically of anionic nanoliposomesconjugated mono with-, di enhanced- and trisaccharides stability [83 and].They the phosphatidyl studied the correlation group with between their stability enzymatically against conjugateddehydration/rehydration mono-, di- and processes. trisaccharides Liposomial and the vesicles phosphatidyl have always group been with considered their stability very against useful dehydrationtools in the /rehydration delivery of processes. drugs, nutrients Liposomial or genes vesicles; however have always, the limited been considered use of PC very as useful a primary tools incon thestituent delivery of new of drugs, liposomes nutrients is linked or genes; to its however, low stability the limited[84]. The use conjugation of PC as a primaryof the phosphatidyl constituent ofgroup new with liposomes different is linkedsugar tomoieties its low with stability an increasing [84]. The conjugationbulky structure of the appeared phosphatidyl to be the group solution with dithatfferent would sugar allow moieties the use with of PC an as increasing a building bulky block structure as it is stabilized appeared by to bethe the presence solution of that the wouldsaccharides. allow theThe usesynthesis of PC asof athese building new blockPLs was as it performed is stabilized in bya biphasic the presence medium, of the where saccharides. PC was The dissolved synthesis in ofethyl these acetate new, whilst PLs was PLD performed from Streptomyc in a biphasices sp. was medium, dissolved wherein sodium PC acetate was dissolved buffer adjusted in ethyl to acetate,pH 5.6. whilstThis buffer PLD fromwas thenStreptomyces added tosp. the was reactor dissolved containing in sodium glucose/sucrose/raffinose acetate buffer adjusted and to pHstirred 5.6. Thisfor 10 bu minffer wasbefore then the added addition to theof the reactor PC solution. containing The glucosereaction/sucrose was left/ra atffi 200nose rpm and at stirred50 °C for for 2 10h, then min beforequenched the additionby the addition of the PCof chloroform. solution. The The reaction yields of was the left different at 200 PLs rpm depended at 50 ◦C for on 2the h, steric then quenchedhindranceby of the additionalcohols: ofPX chloroform.-15 was obtained The yields with of a the95% di ffyield,erent PX PLs-16 depended with 67% on and the PX steric-17 hindrancewith a 23% of yield. the alcohols: These PX-15new PLswas presented obtained with a higher a 95% negative yield, PX-16 surfacewith charge 67% and and PX-17 were with smaller a 23% in sizeyield. compared These new to PLs PC presentednanoliposomes. a higher The negative enhanced surface stabilizing charge and effect, were showed smaller by in sizethese compared nanoliposomes to PC nanoliposomes. based on the Thephosphatidyl enhanced stabilizingsaccharides e ffPXect,-15/16 showed, prevented by these the nanoliposomes aggregation based and fusion on the during phosphatidyl storage saccharides. This was PX-15due to/16 the, prevented presence theof many aggregation OH functionalities and fusion during which storage. resulted This in wasa higher due to electrostatic the presence repulsive of many OHforce functionalities and a higher formation which resulted of H-bonds, in a higher whilst electrostatic the increasing repulsive amount force of sugar and arings higher from formation glucose ofto H-bonds, raffinose whilst increme thented increasing the critical amount micelle of sugar concentration rings from (CMC). glucose The to ra resultsffinose provedincremented that the criticalcompounds micelle PX concentration-14/16 could (CMC). form nanoliposomes The results proved without that thethe compoundsfurther additionPX-14 /16 of could stabilizing form nanoliposomesexcipients, and withoutthey could the be further used additionfor the preparation of stabilizing of excipients, noncytotoxic and and they serum could stable be used deliv forery the preparationsystems. of noncytotoxic and serum stable delivery systems.

Scheme 9. Schematic equation of the saccharides saccharides–phosphatidylderivatives–phosphatidylderivatives (PX-15PX-15/17/17). 2.2.6. 1-Phosphatidyl-β-d-glucose 2.2.6. 1-Phosphatidyl-β-D-glucose 1-Phosphatidyl-β-d-glucose (1-PGlc, PX-18) is a PL which has been discovered in red blood cells 1-Phosphatidyl-β-D-glucose (1-PGlc, PX-18) is a PL which has been discovered in red blood cells of the human umbilical cord and is found in epithelial cells of various human organs, in human of the human umbilical cord and is found in epithelial cells of various human organs, in human neutrophils and in the subventricular zone of the adult mouse brains [85]. In order to study thoroughly neutrophils and in the subventricular zone of the adult mouse brains [85]. In order to study thoroughly the function of this compound, a preparation method was required since the amount of lipid obtained from natural sources was very limited. However, the transphosphatidylation with wild type-PLD from Streptomyces PMF allowed the preparation of 6-phosphatidyl-β-D-glucose (PX- 19) which is the isomer of PX-18, as reported by D’Arrigo et al., because of the higher reaction rate of

Catalysts 2020, 10, 997 12 of 19 the function of this compound, a preparation method was required since the amount of lipid obtained from natural sources was very limited. However, the transphosphatidylation with wild type-PLD from Streptomyces β d PX-19 Catalysts 2020, 10PMF, x FOR allowed PEER REVIEW the preparation of 6-phosphatidyl- - -glucose ( ) which is the isomer12 of of19 PX-18, as reported by D’Arrigo et al., because of the higher reaction rate of the primary hydroxyl group ofthe - βprimary-d-glucose hydroxyl (as shown group in Schemeof -β-D- glucose10)[33]. (as Therefore, shown in more Scheme recently, 10) [33]. in 2016, Therefore, Inoue etmore al. proposedrecently, thein 2016, use of Inoue an engineered et al. proposed enzyme the for use the of same an engineered reaction constituted enzyme byfor athe variant same of reaction PLD from constitutedStreptomyces by 187Ka variant/191W of/ 385YPLD (KWY)from Streptomyces [86]. In detail, 187K/191W/385Y the optimized (KWY) protocol [86]. was In performed detail, the byoptimized dissolving protocol PC in ethylwas performed acetate and by mixing dissolving it with PC ain saturated ethyl acetate solution and mixing of NaCl it with and 50a saturated mM glycylglycine-NaOH solution of NaCl and buff 50er atmM pH glycylglycine 7.5 containing-NaOH KWY-PLD buffer and at pH glucose. 7.5 containing The reaction KWY was-PLD left and shaking glucose. for 22The h reaction at 20 ◦C inwas order left toshaking produce for the22 h desired at 20 °C product in orderPX-18, to producewhich the was desired obtained product with PX a- final18, which yield was of 12.5% obtained due with to the a formationfinal yield ofof undesired12.5% due positionalto the formation isomers of as undesired byproducts. positional isomers as byproducts.

Scheme 10. SchematicSchematic equation equation of of the the synthesis synthesis of of 1 1-phosphatidylglucose-phosphatidylglucose (1 (1-PGlc,-PGlc, 1,2 1,2-dioleoyl--dioleoyl- phosphatidyl-1-glucose,phosphatidyl-1-glucose, PXPX-18-18) and and 6- 6-phosphatidylglucosephosphatidylglucose (6 (6-PGlc,-PGlc, 1,2 1,2-dioleoyl-phosphatidyl--dioleoyl-phosphatidyl-6- 6-glucoseglucose PXPX-19-19). ). 2.2.7. Phosphatidyl-Batyl Alcohol 2.2.7. Phosphatidyl-Batyl Alcohol In 2017, Arranz-Martinez et al. studied the enzymatic transphosphatidylation between PC In 2017, Arranz-Martinez et al. studied the enzymatic transphosphatidylation between PC and and batyl alcohol to produce a novel PL-glyceryl ether, phosphatidyl-batyl alcohol (PBA, PX-20, batyl alcohol to produce a novel PL-glyceryl ether, phosphatidyl-batyl alcohol (PBA, PX-20, see see Scheme 11) with the purpose of obtaining new liposomes [87]. In order to optimize the bioconversion, Scheme 11) with the purpose of obtaining new liposomes [87]. In order to optimize the bioconversion, the authors investigated the following parameters: the type of organic phases (in particular the use the authors investigated the following parameters: the type of organic phases (in particular the use of GRAS organic solvents), the different ratios between aqueous and organic phase, the amount of of GRAS organic solvents), the different ratios between aqueous and organic phase, the amount of calcium chloride, the reaction temperature, the percentage of PLD and the molar concentration of the calcium chloride, the reaction temperature, the percentage of PLD and the molar concentration of the reactants. This study presented a very promising tool that could be applied for the synthesis of highly reactants. This study presented a very promising tool that could be applied for the synthesis of highly valuable ingredients, which can potentially be used as bioactive lipids or as drug delivery vessels. The best results, which allowed the obtainment of 70% of PX-20, was performed by using 5% (w/w) of PLD, an equimolar ratio of reactants and limonene/acetate buffer with a volumetric ratio of 1:3. This process was optimized and scaled up allowing the obtainment of similar results in terms of yield and composition. The concentration of both substrates in the reaction mixture was 126 mmol/L, and the ratio of organic phase (limonene)/sodium acetate buffer 0.1 M at pH 5.5 was 1/3. The incubation

Catalysts 2020, 10, 997 13 of 19 valuable ingredients, which can potentially be used as bioactive lipids or as drug delivery vessels. The best results, which allowed the obtainment of 70% of PX-20, was performed by using 5% (w/w) of PLD, an equimolar ratio of reactants and limonene/acetate buffer with a volumetric ratio of 1:3. CatalystsThis process 2020, 10 was, x FOR optimized PEER REVIEW and scaled up allowing the obtainment of similar results in terms of13 yield of 19 and composition. The concentration of both substrates in the reaction mixture was 126 mmol/L, and the wasratio performed of organic at phase 50 °C (limonene) for 48 h in/sodium the presence acetate of bu 67ffer mM 0.1 CaCl M at2 pH. After 5.5 wascentrifugation 1/3. The incubation, this allowed was theperformed obtainment at 50 of◦C two for 48liquid h in phases the presence and a of solid 67 mM one; CaCl the 2 solid. After interface centrifugation, was washed this allowed twice with the organicobtainment solvent of two and liquid three times phases with and distilled a solid one; water the before solid interfacebeing centrifuged was washed again. twice The with solid organic phase wassolvent then and freeze three-dried times in withorder distilled to obtain water a solid before residue. being With centrifuged this procedure again., The the soliddesired phase product was thenPX- 20freeze-dried was obtained in order(87% yield) to obtain with a a solid purity residue. higher Withthan 70%, this procedure,which allowed the desiredit to be tested product as aPX-20 bioactivewas lipid.obtained (87% yield) with a purity higher than 70%, which allowed it to be tested as a bioactive lipid.

SchemeScheme 11. 11. SchematicSchematic equation equation of of the the synthesis synthesis of of phosphatidyl phosphatidyl-batyl-batyl alcohol alcohol (PBA, (PBA, PXPX-20-20).). 2.2.8. Phosphatidylhydroxybutyrate 2.2.8. Phosphatidylhydroxybutyrate Very recently, in 2019, Li et al. described the preparation of a novel PL, phosphatidylhydroxybutyrate Very recently, in 2019, Li et al. described the preparation of a novel PL, (PB, PX-21), for the application in the field of anesthetic and sedatives [88]. The new PL, was prepared phosphatidylhydroxybutyrate (PB, PX-21), for the application in the field of anesthetic and sedatives by transphosphatidylation of PC with sodium γ-hydroxybutyrate (NaGHB, as shown in Scheme 12). [88]. The new PL, was prepared by transphosphatidylation of PC with sodium γ-hydroxybutyrate NaGHB is a naturally occurring neurotransmitter produced by the central nervous system metabolism (NaGHB, as shown in Scheme 12). NaGHB is a naturally occurring neurotransmitter produced by the and has many applications in the medical field being a psychoactive molecule. GHB is an anesthetic central nervous system metabolism and has many applications in the medical field being a and hypnotic agent that enables patients to physiological sleep. It is employed to treat symptoms of psychoactive molecule. GHB is an anesthetic and hypnotic agent that enables patients to alcohol dependence and opiate withdrawal syndrome. Moreover, it is also employed as an adjuvant physiological sleep. It is employed to treat symptoms of alcohol dependence and opiate withdrawal for diseases such as Parkinson’s and Alzheimer’s. However, NaGHB being a hydrophilic and small syndrome. Moreover, it is also employed as an adjuvant for diseases such as Parkinson’s and molecule has difficulties to pass the blood–brain barrier in order to act on the central nervous system Alzheimer’s. However, NaGHB being a hydrophilic and small molecule has difficulties to pass the and is rapidly metabolized. For those reasons, high dosages of NaGHB should be used in order to be blood–brain barrier in order to act on the central nervous system and is rapidly metabolized. For active, with many adverse side effects. For that reason, the authors decided to attach the NaGHB as the those reasons, high dosages of NaGHB should be used in order to be active, with many adverse side polar head of a new PL aiming to decrease the hydrophilicity of the drug, to increase the solubility in effects. For that reason, the authors decided to attach the NaGHB as the polar head of a new PL lipids and, therefore, obtain an improvement of the mobility through cell membranes. It was noticed aiming to decrease the hydrophilicity of the drug, to increase the solubility in lipids and, therefore, that after the absorption into the human brain of PX-21, the widely present PLD hydrolyzed the polar obtain an improvement of the mobility through cell membranes. It was noticed that after the head and released the drug in loco. Two general synthesis procedures were studied, one using an absorption into the human brain of PX-21, the widely present PLD hydrolyzed the polar head and aqueous–solid system and one a liquid–liquid system. The aqueous–solid procedure started with the released the drug in loco. Two general synthesis procedures were studied, one using an aqueous– dissolution by ultrasounds of PC in ethylacetate, followed by the addition of nanoscale silicon dioxide solid system and one a liquid–liquid system. The aqueous–solid procedure started with the and acetone, and the mixture was left shaking at 400 rpm for 3 h at room temperature. This procedure dissolution by ultrasounds of PC in ethylacetate, followed by the addition of nanoscale silicon dioxide was performed to absorb the PC on the silicon dioxide, which was then collected by centrifugation at and acetone, and the mixture was left shaking at 400 rpm for 3 h at room temperature. This procedure 15 ◦C for 20 min at 3500 g. This precipitate was washed at first with distilled water and suspended in was performed to absorb× the PC on the silicon dioxide, which was then collected by centrifugation at 15 0.2 M of acetate buffer at pH 5.5 containing PLD and NaGHB. The incubation was conducted at 35 ◦C °C for 20 min at 3500× g. This precipitate was washed at first with distilled water and suspended in 0.2 at 400 rpm for 12 h. This method was also compared with a liquid–liquid process where a solution M of acetate buffer at pH 5.5 containing PLD and NaGHB. The incubation was conducted at 35 °C at containing PC in diethylether was mixed with a PLD solution containing NaGHB. This incubation 400 rpm for 12 h. This method was also compared with a liquid–liquid process where a solution was performed at 30 ◦C and 400 rpm. The aqueous–solid system appeared to be much better for containing PC in diethylether was mixed with a PLD solution containing NaGHB. This incubation various reasons: firstly, the conversion rate of PC was close to 100%, and the purity was up to 97%; was performed at 30 °C and 400 rpm. The aqueous–solid system appeared to be much better for secondly, it was an environmentally friendly method which was scaled up from 2 to 250 mL of reaction various reasons: firstly, the conversion rate of PC was close to 100%, and the purity was up to 97%; volume yielding 94% of PX-21 and just 2.3% of PA; and at the end, the free PLD solution was reused secondly, it was an environmentally friendly method which was scaled up from 2 to 250 mL of for different cycles, allowing a yield of 70.7% of PB after the 5th cycle. reaction volume yielding 94% of PX-21 and just 2.3% of PA; and at the end, the free PLD solution was reused for different cycles, allowing a yield of 70.7% of PB after the 5th cycle.

Scheme 12. Schematic equation of the synthesis of phosphatidylhydroxybutyrate (PB, PX-21).

Catalysts 2020, 10, x FOR PEER REVIEW 13 of 19

was performed at 50 °C for 48 h in the presence of 67 mM CaCl2. After centrifugation, this allowed the obtainment of two liquid phases and a solid one; the solid interface was washed twice with organic solvent and three times with distilled water before being centrifuged again. The solid phase was then freeze-dried in order to obtain a solid residue. With this procedure, the desired product PX- 20 was obtained (87% yield) with a purity higher than 70%, which allowed it to be tested as a bioactive lipid.

Scheme 11. Schematic equation of the synthesis of phosphatidyl-batyl alcohol (PBA, PX-20).

2.2.8. Phosphatidylhydroxybutyrate Very recently, in 2019, Li et al. described the preparation of a novel PL, phosphatidylhydroxybutyrate (PB, PX-21), for the application in the field of anesthetic and sedatives [88]. The new PL, was prepared by transphosphatidylation of PC with sodium γ-hydroxybutyrate (NaGHB, as shown in Scheme 12). NaGHB is a naturally occurring neurotransmitter produced by the central nervous system metabolism and has many applications in the medical field being a psychoactive molecule. GHB is an anesthetic and hypnotic agent that enables patients to physiological sleep. It is employed to treat symptoms of alcohol dependence and opiate withdrawal syndrome. Moreover, it is also employed as an adjuvant for diseases such as Parkinson’s and Alzheimer’s. However, NaGHB being a hydrophilic and small molecule has difficulties to pass the blood–brain barrier in order to act on the central nervous system and is rapidly metabolized. For those reasons, high dosages of NaGHB should be used in order to be active, with many adverse side effects. For that reason, the authors decided to attach the NaGHB as the polar head of a new PL aiming to decrease the hydrophilicity of the drug, to increase the solubility in lipids and, therefore, obtain an improvement of the mobility through cell membranes. It was noticed that after the absorption into the human brain of PX-21, the widely present PLD hydrolyzed the polar head and released the drug in loco. Two general synthesis procedures were studied, one using an aqueous– solid system and one a liquid–liquid system. The aqueous–solid procedure started with the dissolution by ultrasounds of PC in ethylacetate, followed by the addition of nanoscale silicon dioxide and acetone, and the mixture was left shaking at 400 rpm for 3 h at room temperature. This procedure was performed to absorb the PC on the silicon dioxide, which was then collected by centrifugation at 15 °C for 20 min at 3500× g. This precipitate was washed at first with distilled water and suspended in 0.2 M of acetate buffer at pH 5.5 containing PLD and NaGHB. The incubation was conducted at 35 °C at 400 rpm for 12 h. This method was also compared with a liquid–liquid process where a solution containing PC in diethylether was mixed with a PLD solution containing NaGHB. This incubation was performed at 30 °C and 400 rpm. The aqueous–solid system appeared to be much better for various reasons: firstly, the conversion rate of PC was close to 100%, and the purity was up to 97%; secondly, it was an environmentally friendly method which was scaled up from 2 to 250 mL of

reactionCatalysts 2020 volume, 10, 997 yielding 94% of PX-21 and just 2.3% of PA; and at the end, the free PLD solution14 was of 19 reused for different cycles, allowing a yield of 70.7% of PB after the 5th cycle.

Catalysts 2020, 10, x FOR PEER REVIEW 14 of 19 SchemeScheme 12. SchematicSchematic equation equation of of the the synthesis synthesis of of phosphatidyl phosphatidylhydroxybutyratehydroxybutyrate (PB, PXPX-21-21). 2.2.9. Phosphatidylpanthenol 2.2.9. Phosphatidylpanthenol In the current year (2020), Yamamoto et al. exploited the PLD-catalyzed transphosphatidylation of PCIn to theprepare current a new year PL, (2020), the phosphatidylpanthenol Yamamoto et al. exploited (PX- the22, PLD-catalyzedsee Scheme 13). transphosphatidylation In fact, D-panthenol is knownof PC to to prepare be a pro a new-vitamin PL, the B5 phosphatidylpanthenol and to possess many interesting (PX-22, see Schemeproperties 13). such In fact, as being D-panthenol wound is- healing,known to anti be- aoxidative pro-vitamin and B5 anti and-inflammatory. to possess many However, interesting its high properties hydrophilicity such as being requires wound-healing, the use of emulsifiers.anti-oxidative The and idea anti-inflammatory. of the authors wa However,s to link itsthe high D-panthenol hydrophilicity as the requires polar head the use of ofa new emulsifiers. PL, in orderThe idea to enhance of the authors its biocompatibility was to link the and D-panthenol to test its anti as the-inflammatory polar head of activity a new on PL, the in ordermacrophagelike to enhance RAW264.7its biocompatibility cell. The and results to test were its very anti-inflammatory interesting showing activity that on the the macrophagelikePX-22 was better RAW264.7 than the cell. D- panthenolThe results from were an very anti interesting-inflammatory showing point thatof view. the PX-22The synthesiswas better was than performed the D-panthenol as follows: from 31 mM an soyPCanti-inflammatory in ethyl acetate point was of incubated view. The with synthesis PLD from was Streptomyces performed assp. follows: in sodium 31 acetate mM soyPC buffer in 0.2 ethyl M atacetate pH 5.6, was 10 incubated mM CaCl with2 and PLD D from-panthenolStreptomyces with sp. a concentration in sodium acetate of 0.625 buff er M. 0.2 The M at incubation pH 5.6, 10 wasmM performedCaCl2 and at D-panthenol 37 °C for 24 with h and a concentrationprovided, after of precipitation 0.625 M. The and incubation extraction, was the performed desired product at 37 ◦C PX for- 2224 with h and a provided,96% yield. after precipitation and extraction, the desired product PX-22 with a 96% yield.

SchemeScheme 13. 13. SchematicSchematic equation equation of of the the synthesis synthesis of of phosphatidylpanthe phosphatidylpanthenolnol (PXPX-22-22).).

3. Conclusions 3. Conclusions The inclusion of a bioactive nucleophile in the PL backbone as the polar head part of the molecule The inclusion of a bioactive nucleophile in the PL backbone as the polar head part of the molecule is a very suitable strategy in order to stabilize and to allow a better release of active alcohols in vivo is a very suitable strategy in order to stabilize and to allow a better release of active alcohols in vivo because the PL residue represents a nontoxic carrier moiety with a high affinity for cell membranes and because the PL residue represents a nontoxic carrier moiety with a high affinity for cell membranes a great hydrophobicity which prevents bioactive moieties degradation. For those reasons, the polar and a great hydrophobicity which prevents bioactive moieties degradation. For those reasons, the head modified PLs (PX-1 to PX-21) reported in this review present distinctive properties, which are polar head modified PLs (PX-1 to PX-21) reported in this review present distinctive properties, which actually exploited in very diverse fields of applications and can be considered as prodrugs. In this are actually exploited in very diverse fields of applications and can be considered as prodrugs. In this mainframe, the development of practical methods for the preparation of the structurally variable polar mainframe, the development of practical methods for the preparation of the structurally variable head-modified PLs is an important prerequisite to achieve the establishment of structure–activity polar head-modified PLs is an important prerequisite to achieve the establishment of structure– relationships of these compounds. A biocatalytic approach for the synthesis of modified PLs is activity relationships of these compounds. A biocatalytic approach for the synthesis of modified PLs preferred when compared to the chemical processes especially due to the very high selectivity and is preferred when compared to the chemical processes especially due to the very high selectivity and the use of safer reagents and milder conditions. The modification of the polar head of PC (the most the use of safer reagents and milder conditions. The modification of the polar head of PC (the most abundant natural PL) is performed by using the PLD from bacterial sources. This enzyme catalyzes abundant natural PL) is performed by using the PLD from bacterial sources. This enzyme catalyzes the hydrolysis of PC to choline and PA, but in the presence of an appropriate acceptor (an alcohol), the hydrolysis of PC to choline and PA, but in the presence of an appropriate acceptor (an alcohol), it performs the transphosphatidylation reaction, which allows the modification of the polar head. it performs the transphosphatidylation reaction, which allows the modification of the polar head. Parameters which may affect the ratios between the production of the new PLs and PA and the overall Parameters which may affect the ratios between the production of the new PLs and PA and the overall production yield depends on the reaction conditions, in particular, the concentration and nature of the production yield depends on the reaction conditions, in particular, the concentration and nature of alcohol, as well as the enzymatic source. Very different strains of PLD have been studied, some of the alcohol, as well as the enzymatic source. Very different strains of PLD have been studied, some which was shown to be highly able to perform transphosphatidylation reactions. The availability of which was shown to be highly able to perform transphosphatidylation reactions. The availability of enzymes which possess a very high substrate specificity and high transfer capacity offers a very of enzymes which possess a very high substrate specificity and high transfer capacity offers a very appealing approach which allows the synthesis of natural and synthetic polar head modified PLs to be appealing approach which allows the synthesis of natural and synthetic polar head modified PLs to performed, without any comparison with chemical approaches. All the bioconversions described in be performed, without any comparison with chemical approaches. All the bioconversions described this review follow a relatively simple and well-established procedure already known for the general in this review follow a relatively simple and well-established procedure already known for the scheme in the middle of the 1990s, which allows the recovery of numerous new products and the general scheme in the middle of the 1990s, which allows the recovery of numerous new products and the investigations on their potential effects both in the medical field as new drug delivery systems, as well as for the cosmetic industry, thanks to their antibacterial and antioxidative activities. This overview will hopefully constitute a source of inspiration for new potential investigations on PLs’ fertile area of research.

Catalysts 2020, 10, 997 15 of 19 investigations on their potential effects both in the medical field as new drug delivery systems, as well as for the cosmetic industry, thanks to their antibacterial and antioxidative activities. This overview will hopefully constitute a source of inspiration for new potential investigations on PLs’ fertile area of research.

Author Contributions: Conceptualization, P.D., writing—original draft preparation, C.A., P.D.; writing—review and editing, C.A., F.D., L.R., P.D.; funding acquisition, P.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fondazione Cariplo—INNOVHUB-SSI, grant n. 2017–1015, Project SOAVE (Seed and vegetable Oils Active Valorization through Enzymes; Bando congiunto Ricerca Integrata sulle Biotecnologie industriali e sulla Bioeconomia ed. 2017). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Nickels, J.D.; Smith, J.C.; Cheng, X. Lateral organization, bilayer asymmetry, and inter-leaflet coupling of biological membranes. Chem. Phys. Lipids 2015, 192, 87–99. [CrossRef][PubMed] 2. Dowhan, W.; Bogdanov, M.; Mileykovskaya, E. CHAPTER 1—Functional roles of lipids in membranes. In Biochemistry of Lipids, Lipoproteins and Membranes, 5th ed.; Vance, D.E., Vance, J.E., Eds.; Elsevier: San Diego, CA, USA, 2008; pp. 1–37. [CrossRef] 3. Vance, D.E.; Vance, J.E. CHAPTER 8—Phospholipid biosynthesis in eukaryotes. In Biochemistry of Lipids, Lipoproteins and Membranes, 5th ed.; Vance, D.E., Vance, J.E., Eds.; Elsevier: San Diego, CA, USA, 2008; pp. 213–244. [CrossRef] 4. Isabel, E.-S.; Arantxa, R.-C.; Susana, M.; Agustin, R.-G.; Jose, M.O.; de Ana Ramirez, M. Beneficial Effects of Bioactive Phospholipids: Genomic Bases. Curr. Nutr. Food Sci. 2011, 7, 145–154. [CrossRef] 5. Falconi, M.; Ciccone, S.; D’Arrigo, P.; Viani, F.; Sorge, R.; Novelli, G.; Patrizi, P.; Desideri, A.; Biocca, S. Design of a novel LOX-1 receptor antagonist mimicking the natural substrate. Biochem. Biophys. Res. Commun. 2013, 438, 340–345. [CrossRef] 6. Niezgoda, N.; Gliszczyñska, A.; Gladkowski, W.; Kempiñska, K.; Wietrzyk, J.; Wawrzeñczyk, C. Phosphatidylcholine with cis-9, trans-11 and trans-10, cis-12 Conjugated Linoleic Acid Isomers: Synthesis and Cytotoxic Studies. Aust. J. Chem. 2015, 68, 1065–1075. [CrossRef] 7. D’Arrigo, P.; Scotti, M. Lysophospholipids: Synthesis and Biological Aspects. Curr. Org. Chem. 2013, 17, 812–830. [CrossRef] 8. Küllenberg, D.; Taylor, L.A.; Schneider, M.; Massing, U. Health effects of dietary phospholipids. Lipids Health Dis. 2012, 11, 3. [CrossRef]

9. Schverer, M.; O0Mahony, S.M.; O’Riordan, K.J.; Donoso, F.; Roy, B.L.; Stanton, C.; Dinan, T.G.; Schellekens, H.; Cryan, J.F. Dietary phospholipids: Role in cognitive processes across the lifespan. Neurosci. Biobehav. Rev. 2020, 111, 183–193. [CrossRef] 10. Baldassarre, F.; Allegretti, C.; Tessaro, D.; Carata, E.; Citti, C.; Vergaro, V.; Nobile, C.; Cannazza, G.; D’Arrigo, P.; Mele, A.; et al. Biocatalytic Synthesis of Phospholipids and Their Application as Coating Agents for CaCO3 Nano–crystals: Characterization and Intracellular Localization Analysis. ChemistrySelect 2016, 1, 6507–6514. [CrossRef] 11. Ishii, F.; Nii, T. Chapter 22—Lipid emulsions and lipid vesicles prepared from various phospholipids as drug carriers. In Colloid and Interface Science in Pharmaceutical Research and Development; Ohshima, H., Makino, K., Eds.; Elsevier: San Diego, CA, USA, 2014; pp. 469–501. [CrossRef] 12. Khan, I.; Elhissi, A.; Shah, M.; Alhnan, M.A.; Ahmed, W. Liposome-based carrier systems and devices used for pulmonary drug delivery. In Biomaterials and Medical Tribology; Davim, J.P., Ed.; Woodhead Publishing: Cambridge, UK, 2013; pp. 395–443. [CrossRef] 13. Li, J.; Wang, X.; Zhang, T.; Wang, C.; Huang, Z.; Luo, X.; Deng, Y. A review on phospholipids and their main applications in drug delivery systems. Asian J. Pharm. Sci. 2015, 10, 81–98. [CrossRef] 14. Yadav, S.; Sharma, A.K.; Kumar, P.Nanoscale Self-Assembly for Therapeutic Delivery. Front. Bioeng. Biotechnol. 2020, 8.[CrossRef] 15. Allen, T.M.; Cullis, P.R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev. 2013, 65, 36–48. [CrossRef] Catalysts 2020, 10, 997 16 of 19

16. Sercombe, L.; Veerati, T.; Moheimani, F.; Wu, S.Y.; Sood, A.K.; Hua, S. Advances and Challenges of Liposome Assisted Drug Delivery. Front. Pharmacol. 2015, 6.[CrossRef] 17. Ozturk, B.; McClements, D.J. Progress in natural emulsifiers for utilization in food emulsions. Curr. Opin. Food Sci. 2016, 7, 1–6. [CrossRef] 18. Koprivnjak, O.; Škevin, D.; Vali´c,S.; Majeti´c,V.; Petriˇcevi´c,S.; Ljubenkov, I. The antioxidant capacity and oxidative stability of virgin olive oil enriched with phospholipids. Food Chem. 2008, 111, 121–126. [CrossRef] 19. Cui, L.; Decker, E.A. Phospholipids in foods: Prooxidants or antioxidants? J. Sci. Food Agric. 2016, 96, 18–31. [CrossRef][PubMed] 20. Zabielska-Koczyw ˛as,K.; Lechowski, R. The Use of Liposomes and Nanoparticles as Drug Delivery Systems to Improve Cancer Treatment in Dogs and Cats. Molecules 2017, 22, 2167. [CrossRef] 21. D’Arrigo, P.; Fasoli, E.; Pedrocchi-Fantoni, G.; Rossi, C.; Saraceno, C.; Tessaro, D.; Servi, S. A practical selective synthesis of mixed short/long chains glycerophosphocholines. Chem. Phys. Lipids 2007, 147, 113–118. [CrossRef] 22. Gładkowski, W.; Chojnacka, A.; Kiełbowicz, G.; Trziszka, T.; Wawrze´nczyk,C. Isolation of Pure Phospholipid Fraction from Egg Yolk. J. Am. Oil Chem. Soc. 2012, 89, 179–182. [CrossRef] 23. Weber, E.J. Compositions of commercial corn and soybean lecithins. J. Am. Oil Chem. Soc. 1981, 58, 898–901. [CrossRef] 24. Robert, C.; Couëdelo, L.; Vaysse, C.; Michalski, M.-C. Vegetable lecithins: A review of their compositional diversity, impact on lipid metabolism and potential in cardiometabolic disease prevention. Biochimie 2020, 169, 121–132. [CrossRef] 25. Klang, V.; Valenta, C. Lecithin-based nanoemulsions. J. Drug Deliv. Sci. Technol. 2011, 21, 55–76. [CrossRef] 26. Gliszczy´nska,A.; Niezgoda, N.; Gładkowski, W.; Switalska,´ M.; Wietrzyk, J. Isoprenoid-phospholipid conjugates as potential therapeutic agents: Synthesis, characterization and antiproliferative studies. PLoS ONE 2017, 12, e0172238. [CrossRef][PubMed] 27. Eibl, H. Synthesis of glycerophospholipids. Chem. Phys. Lipids 1980, 26, 405–429. [CrossRef] 28. Ali, S.; Bittman, R. Facile diacylation of glycidyl tosylate. Chiral synthesis of symmetric-chain glycerophospholipids. J. Org. Chem. 1988, 53, 5547–5549. [CrossRef] 29. D’Arrigo, P.; Servi, S. Synthesis of Lysophospholipids. Molecules 2010, 15, 1354. [CrossRef] 30. Lindberg, J.; Ekeroth, J.; Konradsson, P. Efficient Synthesis of Phospholipids from Glycidyl Phosphates. J. Org. Chem. 2002, 67, 194–199. [CrossRef] 31. Fasoli, E.; Arnone, A.; Caligiuri, A.; D’Arrigo, P.; de Ferra, L.; Servi, S. Tin-mediated synthesis of lyso-phospholipids. Org. Biomol. Chem. 2006, 4, 2974–2978. [CrossRef] 32. Massing, U.; Eibl, H. New optically pure dimethylacetals of glyceraldehydes and their application for lipid and phospholipid synthesis. Chem. Phys. Lipids 1995, 76, 211–224. [CrossRef] 33. D’Arrigo, P.; De Ferra, L.; Piergianni, V.; Selva, A.; Servi, S.; Strini, A. Preparative transformation of natural phospholipids catalysed by phospholipase D from Streptomyces. J. Chem. Soc. Perkin Trans. 1 1996, 2651–2656. [CrossRef] 34. Adlercreutz, P.; Virto, C.; Persson, M.; Vaz, S.; Adlercreutz, D.; Svensson, I.; Wehtje, E. Enzymatic conversions of polar lipids. Principles, problems and solutions. J. Mol. Catal. B Enzym. 2001, 11, 173–178. [CrossRef] 35. Bornscheuer, U.T. Enzymes in Lipid Modification. Annu. Rev. Food Sci. Technol. 2018, 9, 85–103. [CrossRef] [PubMed] 36. Anthonsen, T.; D’Arrigo, P.; Pedrocchi-Fantoni, G.; Secundo, F.; Servi, S.; Sundby, E. Phospholipids hydrolysis in organic solvents catalysed by immobilised phospholipase C. J. Mol. Catal. B Enzym. 1999, 6, 125–132. [CrossRef] 37. Zhang, Z.; Chen, M.; Xu, W.; Zhang, W.; Zhang, T.; Guang, C.; Mu, W. Microbial phospholipase D: Identification, modification and application. Trends Food Sci. Technol. 2020, 96, 145–156. [CrossRef] 38. Kolesnikov, Y.S.; Nokhrina, K.P.; Kretynin, S.V.; Volotovski, I.D.; Martinec, J.; Romanov, G.A.; Kravets, V.S. Molecular structure of phospholipase D and regulatory mechanisms of its activity in plant and animal cells. Biochem. Mosc. 2012, 77, 1–14. [CrossRef] 39. Bargmann, B.O.R.; Munnik, T. The role of phospholipase D in plant stress responses. Curr. Opin. Plant Biol. 2006, 9, 515–522. [CrossRef] Catalysts 2020, 10, 997 17 of 19

40. Subileau, M.; Jan, A.-H.; Dubreucq, E. Chapter 3—Lipases/Acyltransferases for Lipid Modification in Aqueous Media. In Lipid Modification by Enzymes and Engineered Microbes; Bornscheuer, U.T., Ed.; AOCS Press: Urbana, IL, USA, 2018; pp. 45–68. [CrossRef] 41. D’Arrigo, P.; Servi, S. Using phospholipases for phospholipid modification. Trends Biotechnol. 1997, 15, 90–96. [CrossRef] 42. Cerminati, S.; Paoletti, L.; Aguirre, A.; Peirú, S.; Menzella, H.G.; Castelli, M.E. Industrial uses of phospholipases: Current state and future applications. Appl. Microbiol. Biotechnol. 2019, 103, 2571–2582. [CrossRef] 43. Joensuu, M.; Wallis, T.P.; Saber, S.H.; Meunier, F.A. Phospholipases in neuronal function: A role in learning and memory? J. Neurochem. 2019, 153, e14918. [CrossRef] 44. Servi, S. Phospholipases as synthetic catalysts. Top. Curr. Chem. 1999, 200, 127–158. 45. D’Arrigo, P.; de Ferra, L.; Piergianni, V.; Ricci, A.; Scarcelli, D.; Servi, S. Phospholipase D from Streptomyces catalyses the transfer of secondary alcohols. J. Chem. Soc. Chem. Commun. 1994, 1709–1710. [CrossRef] 46. Allegretti, C.; Bono, A.; D’Arrigo, P.; Denuccio, F.; De Simeis, D.; Di Lecce, G.; Serra, S.; Tessaro, D.; Viola, M. Valorization of Corn Seed Oil Acid Degumming Waste for Phospholipids Preparation by Phospholipase D-Mediated Processes. Catalysts 2020, 10, 809. [CrossRef] 47. Carrea, G.; D’Arrigo, P.; Mazzotti, M.; Secundo, F.; Servi, S. On the kinetic mechanism of phospholipase D from Streptomyces SP. in an emulsion system. Biocatal. Biotransform. 1997, 15, 251–264. [CrossRef] 48. Bossi, L.; D’Arrigo, P.; Pedrocchi-Fantoni, G.; Mele, A.; Servi, S.; Leiros, I. The substrate requirements of phospholipase D. J. Mol. Catal. B Enzym. 2001, 11, 433–438. [CrossRef] 49. D’Arrigo, P.; Fasoli, E.; Pedrocchi-Fantoni, G.; Servi, S.; Tessaro, D. Membrane assisted coupled enzyme system for phospholipid modification. Enzym. Microb. Technol. 2005, 37, 435–440. [CrossRef] 50. Secundo, F.; Carrea, G.; D’Arrigo, P.; Servi, S. Evidence for an Essential Lysyl Residue in Phospholipase D from Streptomyces sp. by Modification with Diethyl Pyrocarbonate and Pyridoxal 5-Phosphate. Biochemistry 1996, 35, 9631–9636. [CrossRef] 51. Leiros, I.; Hough, E.; D’Arrigo, P.; Carrea, G.; Pedrocchi-Fantoni, G.; Secundo, F.; Servi, S. Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces sp. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000, 56, 466–468. [CrossRef] 52. Carrea, G.; D’Arrigo, P.; Secundo, F.; Servi, S. Purification and applications of a phospholipase D from a new strain of Streptomyces. Biotechnol. Lett. 1997, 19, 1083–1085. [CrossRef] 53. Carrea, G.; D’Arrigo, P.; Piergianni, V.; Roncaglio, S.; Secundo, F.; Servi, S. Purification and properties of two phospholipases D from Streptomyces sp. Biochim. Et Biophys. Acta Lipids Lipid Metab. 1995, 1255, 273–279. [CrossRef] 54. Liu, X.; Shiihara, M.; Taniwaki, N.; Shirasaka, N.; Atsumi, Y.; Shiojiri, M. Phosphatidylserine: Biology, Technologies, and Applications. In Polar Lipids; Ahmad, M.U., Xu, X., Eds.; Elsevier: San Diego, CA, USA, 2015; pp. 145–184. [CrossRef] 55. Juneja, L.R.; Kazuoka, T.; Goto, N.; Yamane, T.; Shimizu, S. Conversion of phosphatidylcholine to phosphatidylserine by various phospholipases D in the presence of l- or d-serine. Biochim. Biophys. Acta Lipids Lipid Metab. 1989, 1003, 277–283. [CrossRef] 56. D’Arrigo, P.; Cerioli, L.; Chiappe, C.; Panzeri, W.; Tessaro, D.; Mele, A. Improvements in the enzymatic synthesis of phosphatidylserine employing ionic liquids. J. Mol. Catal. B Enzym. 2012, 84, 132–135. [CrossRef] 57. Duan, Z.-Q.; Hu, F. Highly efficient synthesis of phosphatidylserine in the eco-friendly solvent γ-valerolactone. Green Chem. 2012, 14, 1581–1583. [CrossRef] 58. Duan, Z.-Q.; Hu, F. Efficient synthesis of phosphatidylserine in 2-methyltetrahydrofuran. J. Biotechnol. 2013, 163, 45–49. [CrossRef][PubMed] 59. Chen, S.; Xu, L.; Li, Y.; Hao, N.; Yan, M. Bioconversion of Phosphatidylserine by Phospholipase D from Streptomyces racemochromogenes in a Microaqueous Water-Immiscible Organic Solvent. Biosci. Biotechnol. Biochem. 2013, 77, 1939–1941. [CrossRef][PubMed] 60. Yang, S.-L.; Duan, Z.-Q. Insight into enzymatic synthesis of phosphatidylserine in deep eutectic solvents. Catal. Commun. 2016, 82, 16–19. [CrossRef] 61. Qin, W.; Wu, C.; Song, W.; Chen, X.; Liu, J.; Luo, Q.; Liu, L. A novel high-yield process of phospholipase D-mediated phosphatidylserine production with cyclopentyl methyl ether. Process Biochem. 2018, 66, 146–149. [CrossRef] Catalysts 2020, 10, 997 18 of 19

62. Gregoriadis, G. Liposomes for drugs and vaccines. Trends Biotechnol. 1985, 3, 235–241. [CrossRef] 63. Sakdiset, P.; Okada, A.; Todo, H.; Sugibayashi, K. Selection of phospholipids to design liposome preparations with high skin penetration-enhancing effects. J. Drug Deliv. Sci. Technol. 2018, 44, 58–64. [CrossRef] 64. Hossann, M.; Wiggenhorn, M.; Schwerdt, A.; Wachholz, K.; Teichert, N.; Eibl, H.; Issels, R.D.; Lindner, L.H. In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes. Biochim. Et Biophys. Acta Biomembr. 2007, 1768, 2491–2499. [CrossRef] 65. Mazela, J.; Merritt, T.A.; Gadzinowski, J.; Sinha, S. Evolution of pulmonary surfactants for the treatment of neonatal respiratory distress syndrome and paediatric lung diseases. Acta Paediatr. 2006, 95, 1036–1048. [CrossRef] 66. Juneja, L.R.; Hibi, N.; Inagaki, N.; Yamane, T.; Shimizu, S. Comparative study on conversion of phosphatidylcholine to phosphatidylglycerol by cabbage phospholipase D in micelle and emulsion systems. Enzym. Microb. Technol. 1987, 9, 350–354. [CrossRef] 67. Piazza, G.J.; Marmer, W.N. Conversion of Phosphatidylcholine to Phosphatidylglycerol with Phospholipase D and Glycerol. J. Am. Oil Chem. Soc. 2007, 84, 645–651. [CrossRef] 68. D’Arrigo, P.; de Ferra, L.; Pedrocchi-Fantoni, G.; Scarcelli, D.; Servi, S.; Strini, A. Enzyme-mediated synthesis of two diastereoisomeric forms of phosphatidylglycerol and of diphosphatidylglycerol (cardiolipin). J. Chem. Soc. Perkin Trans. 1 1996, 2657–2660. [CrossRef] 69. Schlame, M.; Rua, D.; Greenberg, M.L. The biosynthesis and functional role of cardiolipin. Prog. Lipid Res. 2000, 39, 257–288. [CrossRef] 70. Claypool, S.M.; Koehler, C.M. The complexity of cardiolipin in health and disease. Trends Biochem. Sci. 2012, 37, 32–41. [CrossRef] 71. Calzada, E.; Onguka, O.; Claypool, S.M. Chapter Two—Phosphatidylethanolamine Metabolism in Health and Disease. In International Review of Cell and Molecular Biology; Jeon, K.W., Ed.; Academic Press: Cambridge, MA, USA, 2016; Volume 321, pp. 29–88. 72. Patel, D.; Witt, S.N. Ethanolamine and Phosphatidylethanolamine: Partners in Health and Disease. Oxidative Med. Cell. Longev. 2017, 2017, 4829180. [CrossRef] 73. Sun, N.; Chen, J.; Bao, Z.; Wang, D.; An, B.; Lin, S. Egg Yolk Phosphatidylethanolamine: Extraction Optimization, Antioxidative Activity, and Molecular Structure Profiling. J. Food Sci. 2019, 84, 1002–1011. [CrossRef] 74. Juneja, L.R.; Toru, K.; Tsuneo, Y.; Shoichi, S. Kinetic evaluation of conversion of phosphatidylcholine to phosphatidylethanolamine by phospholipase D from different sources. Biochim. Biophys. Acta Lipids Lipid Metab. 1988, 960, 334–341. [CrossRef] 75. Dippe, M.; Mrestani-Klaus, C.; Schierhorn, A.; Ulbrich-Hofmann, R. Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups. Chem. Phys. Lipids 2008, 152, 71–77. [CrossRef] 76. Nagao, A.; Ishida, N.; Terao, J. Synthesis of 6-phosphatidyl-L-ascorbic acid by phospholipase D. Lipids 1991, 26, 390–394. [CrossRef] 77. Koga, T.; Nagao, A.; Terao, J.; Sawada, K.; Mukai, K. Synthesis of a phosphatidyl derivative of vitamin E and its antioxidant activity in phospholipid bilayers. Lipids 1994, 29, 83. [CrossRef] 78. Takami, M.; Hidaka, N.; Miki, S.; Suzuki, Y. Enzymatic Synthesis of Novel Phosphatidylkojic Acid and Phosphatidylarbutin, and Their Inhibitory Effects on Tyrosinase Activity. Biosci. Biotechnol. Biochem. 1994, 58, 1716–1717. [CrossRef] 79. Takami, M.; Suzuki, Y. Enzymatic Synthesis of Novel Phosphatidylgenipin, and Its Enhanced Cytotoxity. Biosci. Biotechnol. Biochem. 1994, 58, 1897–1898. [CrossRef] 80. Yamamoto, Y.; Hosokawa, M.; Kurihara, H.; Miyashita, K. Preparation of Phosphatidylated Terpenes via Phospholipase D-Mediated Transphosphatidylation. J. Am. Oil Chem. Soc. 2008, 85, 313. [CrossRef] 81. Casado, V.; Reglero, G.; Torres, C.F. Production and Scale-up of phosphatidyl-tyrosol catalyzed by a food grade phospholipase D. Food Bioprod. Process. 2013, 91, 599–608. [CrossRef] 82. Di Benedetto, R.; Varì, R.; Scazzocchio, B.; Filesi, C.; Santangelo, C.; Giovannini, C.; Matarrese, P.; D’Archivio, M.; Masella, R. Tyrosol, the major extra virgin olive oil compound, restored intracellular antioxidant defences in spite of its weak antioxidative effectiveness. Nutr. Metab. Cardiovasc. Dis. 2007, 17, 535–545. [CrossRef] Catalysts 2020, 10, 997 19 of 19

83. Song, S.; Cheong, L.-Z.; Falkeborg, M.; Liu, L.; Dong, M.; Jensen, H.M.; Bertelsen, K.; Thorsen, M.; Tan, T.; Xu, X.; et al. Facile Synthesis of Phosphatidyl Saccharides for Preparation of Anionic Nanoliposomes with Enhanced Stability. PLoS ONE 2013, 8, e73891. [CrossRef] 84. Torchilin, V.P. Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Discov. 2005, 4, 145–160. [CrossRef] 85. Nagatsuka, Y.; Kasama, T.; Ohashi, Y.; Uzawa, J.; Ono, Y.; Shimizu, K.; Hirabayashi, Y. A new phosphoglycerolipid, ‘phosphatidylglucose’, found in human cord red cells by multi-reactive monoclonal anti-i cold agglutinin, mAb GL-1/GL-2. FEBS Lett. 2001, 497, 141–147. [CrossRef] 86. Inoue, A.; Adachi, M.; Damnjanovi´c, J.; Nakano, H.; Iwasaki, Y. Direct Enzymatic Synthesis of 1-Phosphatidyl-β-D-glucose by Engineered Phospholipase D. ChemistrySelect 2016, 1, 4121–4125. [CrossRef] 87. Arranz-Martínez, P.; Casado, V.; Reglero, G.; Torres, C.F. Novel glyceryl ethers phospholipids produced by solid to solid transphosphatidylation in the presence of a food grade phospholipase D. Eur. J. Lipid Sci. Technol. 2017, 119, 1600427. [CrossRef] 88. Li, B.; Wang, J.; Li, H.; Zhang, X.; Duan, D.; Yu, W.; Zhao, B. Efficient and green aqueous solid system − for transphosphatidylation to produce phosphatidylhydroxybutyrate: Potential drugs for central nervous system’s diseases. Biotechnol. Prog. 2019, 35, e2726. [CrossRef][PubMed]

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