Investigation of Synthesis of Functionally Substituted Endiines and Their Chemical and Microbiological Conversion

The methods of preparation of endiine and endiallene diols by interaction of cis-1,4-dibrombutene and cis-1,4-dichlorrbutene with monosubstituted acetylene alcohols in presence of the catalytic systems consisting of one-iodide copper, triethylamine and K2СО3 in a medium of dimethylformamide have been developed. It has been shown that unlike 1,4-dibrombutene, the nucleophilic substitution reaction with 1,4-dichlorbutene proceeds by acetylene-allene isomerization with formation of endiallene diols. It has been established that the endiine diols can be used in thin organic synthesis (in the reactions of oxidation, splitting, dehydration, epoxidation, hydrolysis, 1,2-cycloaddition and hypochlorination) with the aim of preparation of practically useful substances. It has been revealed during hydrolysis of epoxide compounds by the chemical and microbiological methods that in the course of microbiological hydrolysis (Aspеrgillus niger), the optically active trans-structured diols are formed.


Results and Discussion
The investigations showed that in interaction of cis-1,4dibrombutene with monosubstituted acetylene alcohols (dimethylethynylcarbinol and propargyl alcohol) in the presence of catalytic system consisting of one-iodide copper, triethylamine and K 2 СО 3 in a medium of dimethylformamide, by the substitution reaction at 55˚C -60˚C the unconjugated endiine diols (I, II) with yields of 65.6% and 74.2% are respectively formed: it has been revealed that unlike 1,4-dibrombutene the nucleophilic substitution reaction with 1,4-dichlorbutene proceeds by acetylene-allene isomerization with formation of endiallene diols (III, IV) with yields 60.4% and 70.1% on Scheme 1.
In the IR-spectra of the compounds (III, IV) along with the absorption bands characteristic for С=С and О−Н bonds, there are also the bands in the field of 1940 -1960 cm −1 , characteristic for >С=С=С< fragment.In the PMR-spectrum of the compounds (III, IV) the protons of СН=С=СН bonds are appeared as a multiplet with chemical shift δ = 5.10 -5.55 ppm.
It has been established that the synthesized diols are very reactive compounds and can undergo the reactions of oxidation, splitting, dehydration, epoxidation, hydrolysis, 1,2-cycloaddition and hypochlorination with formation of new derivatives of diines and endiines.
During carrying out of the oxidation reaction of endiine diol (I) at 35˚C -40˚C by means of oxidative mixture consisting of aqueous solution of chromium anhydride and sulphuric acid the corresponding unsaturated dicerboxylic acid (V) with yield 76.6% is formed (Scheme 2).
In the IR-spectrum of the synthesized compound (V) with presence of strong absorption band at 1725 cm −1 (С=О), the intensive wide band in the field of 2600 -3000 cm −1 , belonging to hydroxyl group has been also detected.
The endiine diol (II) in the presence of powdered potassium hydroxide is subjected to Favorsky reaction accompanying by acetone detachment and is easily converted into endiine (VI) with two terminal acetylene bonds.The dehydration of diol (II) by interaction of potassium bisulphate realized by heating of mixture of reagents in a medium of toluene at 70˚C -80˚C for 6 h leads to the formation of polyunsaturated compounds (VI, VII) with yields 78.6% and 65.4% respectively (Scheme 3).
The structure of the synthesized compounds (VI, VII) has been established on the basis of data of the IR-and PMR-spectra.
In the IR-spectrum of the compound (VI) along with presence of the absorption band at 685 and 1655 cm −1 , characteristic for double НС=СН bond, there is also a band at 2125, 3295 cm −1 , characteristic for terminal acetylene bond.In the IR-spectrum of the compound (VII) along with the absorption bands characteristic for С≡С bonds the band at 2240 cm −1 , characteristic for non-end acetylene bond has been also detected.In this case the absorption bands characteristic for OH group are absent.
In the NMR 1 Н-spectrum of the compound (VI) along with the signals characteristic for cis-НС=СН fragment it has been also identified the signal of proton (2Н, С≡СН) as a triplet at  = 2.43 ppm.
It has been established that the synthesized diol (II) undergoes the epoxidation reaction due to double bond by beans of acetyl hydroperoxide (peracetic acid) forming a diol of diine series with oxirane ring (VIII) with yield 76.5%.Dichlorcyclopropanation of diol (II) proceeds in conditions of interphase catalysis (triethylbenzylammonium chloride, 50% NaOH, chloroform, 20˚C -25˚C) with formation of the corresponding dichlorcyclopropane (IX) on Scheme 4.
In the IR-spectra of the prepared compounds (VII-X) the absorption bands characteristic for С≡СН fragments are absent.In the IR-spectra of the compounds (VIII, IX) along with the absorption bands characteristic for С≡С and О-Н bonds the absorption bands at 3065, 1250 and 955 cm −1 , characteristic for oxirane ring are also present.In the IR-spectrum of the compound (X) the absorption bands at 765, 3085 and 2235 cm −1 , characteristic for valence vibrations of bonds of С-Сl and С-Н methylene groups of three-membered carbon-carbon ring are present.
It has been shown (Scheme 5) that the endiine (IX) undergoes the oxidation reaction with peracetic acid at 20˚C -25˚C on double bond forming an epoxide with two terminal acetylene bonds (XI).The diine epoxide (XI) has been also synthesized by counter synthesis-splitting of epoxydiindol (IX) on Favorsky reverse reaction: the reaction proceeds (yield 68.6%) in the presence of potas-Scheme 3. Synthesis of endiine hydrocarbons.
sium hydroxide in a medium of toluene at 105˚C -110˚C.
lacquer coating is one of the frequently occurring cases of biodeteriorations.Among microorganisms damaging lacquer coatings the fungi of following genera: Аsреrgillus, Penicillium, Fusarium, Trioderma, Alternaria, Серhalosporium, and bacteria-Pseudomonas, Flavobacterium are frequently occurred.The damages of coatings by fungi occur either due to components entering in compositions of coating or due to substances polluting surface of coating, under action of metabolites isolated by mycelium which grows due to substances polluting coating [28].A fungus-resistance of these coatings is decreased in the following series: epoxide, polyurethane, melamine-alkide, organosilicon, pentaphthalic.It has been shown that the isolated strains Aspеrgillus and Fusarium carried out the biohydrolysis of the epoxide compounds of diacetylene series for preparation of products of thin synthesis with the aim of their practical application and prevention of biodeterioration process.
The structure of the synthesized epoxydiine (XI) has been confirmed by data of the IR-and PMR-spectra.
It has been established that the endiine diols (I, II) react at 35˚C -40˚C with HOCl in statu nascendi (in situ) in the induced system HCl + H 2 O 2 , leading to the formation of the corresponding chlorohydrins (XII, XIII).The synthesized chlorohydrins (XII, XIII) are easily dehydrochlorinated in the presence of powdered potassium hydroxide.In this case the oxirane with two terminal acetylene bonds (VIII, XI) with yields 85% -90% is formed (Scheme 6).
The structure of the synthesized chlorohydrins (XII, XIII) has been established by determination of elemental analysis, physical-chemical constants, IR-and NMR 1 Н spectral data.In the IR-spectra of the synthesized chlorohydrins (XII, XIII) the absorption bands in the fields of 3360 -3450, 2225 -2245 and 750-600 cm −1 , characteristic for bonds of О-Н, С≡С and С-Сl respectively have been detected.In this case the absorption bands of valence vibrations of С=С bonds in the field of 1630 -1650 cm −1 are absent.
The maximum degree of biohydrolysis observed in strain Aspergillus niger 15.The analysis of biohydrolysis products of epoxides of acetylene series by the chroma-  In the IR-spectrum of the compounds there are the bands characteristic for terminal acetylene bond (2140, 3300 cm −1 ) and hydroxyl group (3450 cm −1 ).In this case the band characteristic for oxirane ring is absent.
As it was known a leading role in the processes of biodeterioration of the chemical nature construction materials belongs to microscopic fungi (micromycetes) [2].This property of fungi is related to their ability for quick growth and capacity of their enzymatic apparatus.A result of growth of micromycetes on surface of construction materials is decreasing of physical-mechanical and operational characteristics of materials and deterioration of their external view.The microbiological damage of tography and spectral methods showed that during microbiological hydrolysis of epoxide compounds by strains Aspеrgillus niger unlike chemical one of the reaction proceeds with formation of optically active diol with transstructure (XIV) as it was presented at the Scheme 8.
The physical-chemical and spectral data of diol (IХ) obtained by microbiological hydrolysis correspond to data prepared by chemical method.It has been established during implementation of the investigations by the chemical and microbiological methods of hydrolysis of the epoxide compounds of diacetylene series that unlike chemical method of hydrolysis by microbiological method the optically active diols with diterminal acetylene bond are formed (XIV).
It has been established that the biological oxidation of diol (XIV) using Fusarium flocciferum mycelium proceeds with formation of the corresponding diacetylene ketone (XV) and presented on the following Scheme 9.
The results of the mycological investigations showed that the micromycetes can be used as the chemical reagents for preparation of practically useful compounds and the investigation of enzymes causing oxidation will facilitate the development of methods of prevention of biodeteriorations' processes.
Thus, it has been established that based on the high reactivity the prepared functionally substituted endiines can be widely used as the syntones in different syntheses for preparation of practically useful products and intermediate products.

Experimental Material
The IR-spectra of the synthesized compounds were taken on spectrophotometer UK-20 in thin layer.The PMR- spectra were recorded on spectrophotometer Tesla BS-487B (80 MHz), as the internal standard was used hexamethyldisiloxane, solvent-CCl 4 .
The microbiological experiments were conducted using microscopic fungi isolated from oil polluted soil of Absheron.For study of ability of the micromycetes to degrade selected chemicals of the most active strainsdegrader from the following genera: Aspеrgillus, Fusarium, Mucor, Penicillium was used.

Synthesis of diol of diallene series (III, IV)
Oct-4-ene-2,7-diine (VI).In nitrogen current (110˚C -115˚C) it has been distilled 5.5 g (0.025 mol) compound (II) in the presence of 1g powdered potassium hydroxide.The heating was carried out so that the splitting products were slowly distilled to coil receptor.It has been prepared the equimolar mixture of acetone and compound (VI) (in ratio 52:48 on GLC) with m. p. 98˚C -99˚C,

Dichlorcyclopropanation of the compound (II).
To a mixture of 50 ml of 50% aqueous solution of sodium hydroxide and 0.4 g triethylbenzylammonium chloride in mixing for 4 h the solution of 6.6 g (0.03 mol) of the compound (II) in 50ml chloroform was added.The reaction temperature was kept equal to 22˚C -25˚C.The mixture was mixed still for 1h at 40˚C, then was added 200 ml water and extracted by ether.The organic layer was separated, dried over MgSO 4 .After recrystallization the compound (X) was isolated with m. p. 90˚C -91˚C, yield 28.5 %.Found, %: С 59.58, Н 6.52, Cl 23.22.С 15 Н 20 Cl 2 О 2 .Calculated, %: С 59.41, Н 6.65, Cl 23.39.

Synthesis of chlorohydrins of diine series (XII, XIII). General technique:
To a mixture of 0.05 mol of 10% aqueous solution of HCl for preparation of chlorohydrin and 0.05 mol of the compound (I or II) in mixing through dropping funnel 0.06 mol of 26% -30% aqueous solution of hydrogen peroxide (feeding rate 10 g/h) was introduced and was continued the mixing for 5 -6 h at 35˚C -40˚C.After neutralization and drying of organic layer by distillation in vacuum the compounds (XII, XIII) with the following physical-chemical characteristics were isolated: (XII), M. p. 126˚C -127˚C, yield 69.2%.Found, %: С 55.36, Н 6. Counter synthesis of epoxide (XI).To a mixture 13.6 g (0.05 mol) chlorhydrin (XIII) and 25 ml toluene in mixing 10 g powdered potassium hydroxide keeping temperature of reaction mass in the ranges of 40˚C -50˚C was gradually added, then in mixing was heated to 95˚C -100˚C and was endured at this temperature for 8 h.After cooling the organic layer was separated and dried by sodium sulphate.By distillation in the presence of hydroquinone the epoxide (XI) with yield 86.7% with identical data was isolated.
By analogous method from chlorhydrin (XII) the epoxide (VIII) with yield 89.2% with identical data has been prepared.
Chemical hydrolysis of epoxide (XI).6 g (0.05 mol) of 4,5-epoxyocta-1,7-diine (XI) was gradually added to 20 ml of 10% aqueous solution of sulphuric acid.In view of considerable isolation of heat the reaction flask was cooled and in the course of reaction by ice water.After half-hourly mixing the reaction finished.The aqueous solution was saturated by common salt and was repeatedly extracted by ether and then chloroform.After distillation of solvent it was isolated a glycol (XIV) with m. p. 85˚C -86˚C, yield 86.7% by recrystallization.Found, %: С 69.40, Н 7.49.С 8 Н 10 О 2 .Calculated, %: С 69.54, Н 7.30.

Microbiological hydrolysis of epoxide (XI).
1) Preparation of biomass.For preparation of biomass of fungi 3 liter fermenter which filled by 1 litre liquid nutrient medium was used.10ml of liquid paraffin and 0.005 ml of silicon antifoamer for prevention of mass outflow was added to a medium.The incubation was carried out under 25˚C -27˚C.Then the suspension of mycelium of the investigated strains of fungi was added.After two days the incubation of mycelium was filtered, washed with sterile water and placed back in fermenter which already filled by 1l рН 7 phosphate buffer (0.1 М) solution and also medium of enriched nitrogen and air.The yield of product provided by stepwise treatment of samples: for each sample the mycelium was filtered and the obtained biomass of fungi was used in further experiments for realization of biohydrolysis.After decantation the liquid phase was treated by solution NaCl and then was extracted twice by ether.The organic layer was dried (MgSO 4 ), then was steamed in vacuum.
Oxidation of diol (XIV) with use of mycelium Fusarium flocciferum.The crystalline diol (XIV) crushed to powdered mass which was added to flask (2%) containing Czapek's medium without sugar (100 ml) was used for process of oxidation.The biomass was prepared according to the above-mentioned method and in a quantity 5% was added to a medium.The flasks incubated for 5 -7 days at temperature 25˚C -26˚C.After incubation the prepared biomass was filtered, and the remaining liquid was extracted in petroleum-ether after recrystallization was isolated (XV) with m. p. 61˚C -62˚C, yield 78.9%.Found, %: С 71.80, Н 4.40.С 8 Н 6 О 2 .Calculated, %: С 71.63, Н 4.51.

Conclusions
On the basis of splitting reaction (Favorsky reverse reaction) and dehydration of endiine tertiary alcohol, the endiine hydrocarbons being of interest for preparation of analogs of the natural compounds have been synthesized.The synthesized epoxide derivatives of diacetylene series can be used as the chemically active reagents for polymer materials.
The prepared diketones with two terminal acetylene bonds by the microbiological oxidation from the corresponding diols being potential biologically active substances can be used in organic chemistry with the aim to study theoretical and practical problems.It has been revealed during hydrolysis of epoxide compounds by the chemical and microbiological methods that during microbiological hydrolysis, the optically active trans-structured diols are formed which are practically useful substances.
epoxydiine (XI) by the chemical and microbiological methods is of also interest.The hydrolysis reaction (XI) on oxirane ring in the presence of acidic catalysts has been carried out.It has been established that in the presence of 10% aqueous solution of sulphuric acid the epoxydiine (XI) is subjected to the hydrolysis on oxirane ring and in this case the corresponding diol (XIV) with high yield (86.7%) is formed (Scheme 7).