Novel preparation method of carboxylic acid protecting group 2-nitro-phosphoryloxy benzyl compound capable of being sheared by photocatalysis and amino acid derivative of novel carboxylic acid protecting group 2-nitro-phosphoryloxy benzyl compound

Abstract

The invention provides a preparation method of a novel carboxylic acid protecting group capable of photocatalytic shearing and an amino acid derivative thereof, and solves the problems that an existing amino acid protecting group and solid-phase resin are easy to damage or decompose in a deprotection or shearing step and cannot be directly recycled and reused and the like. The preparation method comprises the following steps: (1) reacting (R) 2POCl or POCl3 with coumarin methyl halide containing hydroxyl on a benzene ring, 2-nitrobenzyl halide or alpha-halogenated acetophenone in an alkaline solution, and separating and purifying to obtain a corresponding phosphoryloxy derivative; 2) taking the phosphoryloxy derivative obtained in the step 1) as a raw material, performing hydrolysis or conversion synthesis on a corresponding halogen functional group, and performing separation and purification to obtain a corresponding derivatization product, namely a phosphorus-containing substituted methanol compound; and 3) carrying out condensation coupling on the phosphorus-containing substituted methanol compound obtained in the step 2) and an amino acid (Fmoc-AA-OH) protected by Fmoc or an amino acid (Boc-AA-OH) protected by Boc, and carrying out separation and purification to obtain a corresponding Fmoc-amino-acid ester derivative product of the compound or a Boc-amino-acid ester derivative product of the compound.

Description

[0001] This application is a divisional application. The application number of the original application is 202210218227.8, the application date is March 3, 2022, and the name of the invention is "Method for preparing a novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivatives". Technical Field

[0002] The invention belongs to a method for synthesizing polypeptides in the field of bioorganic chemistry, and specifically relates to a method for preparing a novel carboxylic acid protecting group capable of photocatalytic shearing and an amino acid derivative thereof. Background Art

[0003] Since Theodor Curtius and H. Kolbe reported the synthesis of benzoylglycylglycine in 1882, [1] Since (the first synthesis of N-protected dipeptides), research on peptide synthesis has continued. Scientists have developed N-terminal protecting groups for amino acids such as benzyloxycarbonyl, tert-butyloxycarbonyl (Boc protecting group), and fluorenylmethyloxycarbonyl (Fmoc protecting group). At the same time, various methods for forming peptide bonds have also been studied, such as the acyl chloride method and the azide method to activate the carboxyl group. [2] , activated lipid method and mixed anhydride method [3] .

[0004] 1963 is undoubtedly a memorable year for peptide synthesis. Merrifield creatively pioneered the solid phase synthesis method (SPPS) of peptides. [4] By fixing the N- or C-terminus of the amino acid on the resin, a heterogeneous reaction is performed, which greatly improves the efficiency of peptide synthesis. After a series of amino acids are attached through step-by-step coupling, the peptide chain is sheared off the resin with acid or base to obtain the target product.

[0005] The conditions for releasing peptides by acid-base cleavage are quite harsh. The strong acids, strong bases or strong nucleophiles used may destroy some fragile peptide sequences, thereby affecting the release of the target peptide. [5] Photo-shearing can avoid these problems. By irradiating the "carrier-target product", the target molecule can be gently released (see Figure 1 In 1973, Rich and Gurwara first reported a photosensitive solid phase resin carrier of o-nitrobenzene and released a tripeptide with a yield of 62%. [6] .

[0006] Although photosensitive carriers have such great advantages, their application and progress in peptide synthesis have been unsatisfactory in recent decades, and most of the photosensitive carriers used for peptide synthesis are solid phase carriers. Although many scientists have been studying the photolysis mechanism of small molecule photosensitive protective groups, there are few reports on the application of small molecule photosensitive protective groups in peptide synthesis. Therefore, it is undoubtedly of great significance to study the small molecule photosensitive protective groups of amino acids and their application in peptide synthesis.

[0007] There are currently two main methods for synthesizing peptides using chemical methods: solid phase synthesis and liquid phase synthesis. The advantages and disadvantages of these two methods will not be discussed for now. Regardless of which method is used for peptide synthesis, in order to prevent amino acids from coupling themselves in the presence of condensing agents, the non-reactive groups of amino acids are usually protected. That is, the C-terminus or N-terminus of the amino acid is protected, among which C-terminus (carboxyl) protection is more common, and is generally protected in the form of esters. Therefore, what is finally obtained are peptides with protected carboxyl termini (in the form of esters), and another step of hydrolysis is required to obtain the target peptide. [7] .

[0008] Usually, the hydrolysis of esters is carried out under strong acid or strong base conditions. Some special amino acids or peptides may be destroyed in the shearing process, such as beauvericin and its analogs. Peptides containing ester bonds in the molecule, such as beauvericin, cannot be sheared by traditional shearing methods. Therefore, it is necessary to study a shearing method suitable for such molecules. [8] .

[0009] In 1973, Rich and Gurwara first reported a photosensitive resin (see Figure 2 ) [6] , achieving photo-cleavage of a tripeptide under 350nm light. Subsequently, scientists studied and designed various types of photosensitizing solid-phase resins, and the number of peptides synthesized using photosensitive resins has grown from 3 amino acid residues to more than 10. Although scientists have used some small-molecule photosensitive protecting groups to study the process and mechanism of photolysis reactions, at present, photosensitive protecting groups that can be applied to the total synthesis of peptide chains are limited to solid-phase photosensitive resin carriers. Therefore, in order to achieve more gentle shearing and release peptides under specific circumstances, it is promising to design and synthesize a series of small-molecule amino acid photosensitive protecting groups and verify their application in peptide synthesis.

[0010] Photosensitive protecting groups (PPGs) are also known as caged compounds in biology, which can achieve reagent-free controlled release of substrates. [9] As an ideal photosensitive protective group, it should meet the following conditions: ① The absorption wavelength should be greater than 300nm

[10] (This is suitable for biological applications); ② Stable in dark environments or weak light sources

[10] (to facilitate the synthesis of compounds); ③ suitable substrate release rate (to facilitate the study of its photolysis kinetics). Photosensitive protecting groups have been used in biochemistry, organic synthesis and high-density probes.

[11] It has been applied in some aspects. Next, we will introduce the following common photosensitive protecting groups and their applications in peptide synthesis.

[0011] 1. Coumarin-based photosensitive carrier

[0012] Coumarin and its derivatives are compounds with benzopyrone structure

[12] , which is widely found in some Chinese herbal medicines. Due to its high biocompatibility, high quantum yield and stability

[13] , coumarin and its derivatives have attracted extensive attention from researchers. In recent years, there have been many reports on coumarin and its derivatives used as carboxyl terminal protecting groups.

[0013] The earliest research on coumarin derivatives as photosensitizers can be traced back to 1984, when Givens studied the photocleavage reaction of the conjugate of 7-methoxy-4-methylcoumarin and diethyl phosphate.

[14] When Nu is methanol / piperidine / cysteine, diethyl phosphate can be released by light.

[0014] 4-Coumarinylmethanol derivatives are common precursors of coumarin-caged compounds, which can easily form corresponding esters with carboxylic acids (including amino acids), phosphoric acid and sulfonic acid derivatives. At the same time, some derivatives of the compounds can also serve as protective groups for thiol and amino groups.

[15] .

[0015] As the most successfully applied and studied photosensitive protecting groups, the mechanism of light-releasing carboxylic acids, phosphoric acids and sulfonic acids from coumarin photosensitive protecting groups has been studied thoroughly. Schade and Hagen et al. studied coumarin-phosphate esters and used H2 18 O is photocleaved to obtain coumarin-based methanol and phosphoric acid. 18 O participates in the formation of coumarinylmethanol

[16] The general reaction process can be summarized as follows: coumarin-phosphate ester is cleaved into coumarin-based carbon cation and phosphate by light of appropriate wavelength, and then generates phosphoric acid and coumarin-Nu in the presence of nucleophilic reagent Nu-H (Nu can be RO, RS, etc.).

[0016] In general, according to the data summarized by Petr et al., the maximum absorption wavelength of coumarin-based photosensitive protective groups is roughly distributed between 320-400nm.

[17] , which is consistent with the properties of the good photosensitive protecting groups mentioned above. So far, scientists have conducted many studies on the photolysis mechanism of coumarin photosensitive protecting groups and have made great breakthroughs. However, there are few reports on using coumarin as a photosensitive protecting group for carboxyl compounds (such as amino acids) and applying it to total synthesis. This application will introduce a photosensitive protecting group for amino acids based on coumarin.

[0017] 2. o-Nitrobenzyl carrier

[0018] Although ONB has some disadvantages, for example: ① the excitation wavelength is short and the experimental conditions are harsh; ② the photolysis process is relatively complicated and generally divided into several steps; ③ photolysis generally produces benzaldehyde substances, which are toxic.

[18] However, these do not prevent o-nitrobenzyl protecting groups from becoming the most popular nitrobenzene protecting groups. Most of the photosensitive protecting groups used in various cage compound experiments are o-nitrobenzyl protecting groups.

[0019] The o-nitrobenzyl gene is known for its unique photochemical properties. It was first reported in 1966 by Barltrop et al. who conducted photochemical studies on o-nitrobenzyl esters (carboxylates).

[19] . After illumination, the substrate releases carboxylic acid, and the original o-nitrobenzyl group is converted into an o-nitrosoaldehyde / ketone compound. According to the explanation of the authors and others, they believe that the oxygen atom on the nitro group is transferred during illumination to form a structure similar to acetal, and then the CO bond is broken to release the carboxylic acid anion, and the carrier forms a carbon cation, which is then rearranged to obtain a nitrosoaldehyde / ketone compound. Although the photo-cleavage yield is only 17% and the mechanism is not very clear, this work has laid the foundation for subsequent scientific researchers to study such carriers.

[0020] Since ONB carriers were first reported in 1966, many scientists have been devoted to studying the photolysis mechanism of such carriers. In 2000, Il”Ichev and his colleagues conducted an analytical experiment on the photo-shearing of o-nitrobenzyl ether by time-resolved spectroscopy and other techniques, and made some corrections to the experimental conclusions summarized by previous scientists.

[20] First, after exposure to light, the π electrons on the benzene ring of o-nitrobenzyl ether migrate to generate an intermediate numbered 51, which then undergoes cyclization to generate an intermediate 50, and then the NO bond breaks to generate a hemiacetal intermediate 52, and finally a nitrosoaldehyde compound is generated and methanol is released.

[20] After years of research, the photo-shearing mechanism of ONB carriers has been studied thoroughly.

[0021] As mentioned above, in 1973, Rich and Gurwara successively carried out chloromethylation and nitration reactions on solid phase resin to prepare o-nitrobenzyl photosensitive solid phase resin carrier. A tripeptide was synthesized through this solid phase resin, and the target peptide chain was released under 365nm light, with a release yield of 62%. [6] However, this resin that directly performs multi-step preliminary reactions on the solid phase resin cannot be used for the synthesis of long-chain peptides (it is difficult to directly perform chloromethylation and nitration reactions on the resin). Through this strategy, only tetrapeptides can be synthesized at most. This defect limits the application of ONB photosensitive solid phase carriers in peptide synthesis.

[0022] Subsequently, researchers devoted themselves to the development of ONB-type photosensitive resin carriers and found that the ONB solid-phase photosensitive carrier prepared by first preparing a small molecule with an o-nitrobenzyl structure and then reacting it with a solid-phase resin can overcome the above defects. Rich et al. first prepared the small molecule 3-nitro-4-bromomethylbenzoic acid, then reacted it with an amino-containing resin to prepare an improved ONB-type photosensitive solid-phase resin carrier and completed the synthesis of a decapeptide.

[21] This provides a new idea for the preparation and application of ONB-based carriers, and the subsequent design and synthesis of ONB-based photosensitive resin carriers mostly follow this method.

[0023] The photo-shearing mechanism of ONB-type photosensitive resin carriers is similar to that of small molecule ONB carriers. Although the application in solid phase synthesis is very broad, there are very few small molecule ONB carriers and their applications in liquid phase synthesis, not to mention their applications in peptide synthesis. According to the literature, o-nitrobenzyl-type photosensitive resin carriers play a huge role in the solid phase synthesis of peptides. Therefore, it can be inferred that small molecule ONB can also be used as a photosensitive protective group for amino acids and applied to the liquid phase synthesis of peptides.

[0024] 3. Benzoylmethyl carrier

[0025] In addition to the above two photosensitive carriers, another carrier based on phenacylmethyl [PhC(O)CH2-] is also worthy of further study. Aromatic ketone compounds are generally thermally stable and easy to prepare, which provides them with advantages as photosensitive carriers.

[22] .

[0026] Generally speaking, the carbonyl group of aromatic ketones is the core of photochemical reactivity, while the α-substituted esters of phenacyl groups are the main framework for releasing carboxylic acids.

[23] In 1973, Sheehan et al. proposed a hypothesis about the photolysis mechanism of benzoyl-based carriers by studying the photochemical reaction of p-methoxyphenacyl ester. Using ethanol, 1-4-dioxane and cumene as reaction solvents, they irradiated p-methoxyphenacyl ester with ultraviolet light and finally obtained p-methoxyacetophenone and free carboxylic acid. In addition, acetaldehyde and 2,3-dimethyl-2,3-diphenylbutane (two cumene free radicals can form this compound) were detected in ethanol and cumene as reaction solvents.

[23] .

[0027] Based on the experimental results, Sheehan et al. believed that the CO bond of the benzoyl ester compound was homolytically cleaved after ultraviolet light exposure to generate benzoyl methyl radicals and carboxyl radicals, and then these two radicals captured H atoms from the system to generate acetophenone compounds and free carboxylic acids, respectively. This conclusion is only a guess based on experimental results. At present, there is no experimental data to prove that the photochemical reaction process of this type of carrier is carried out in accordance with homolytic cleavage into two free radicals. With the development of characterization technology, the photochemical mechanism of benzoyl methyl carriers has gradually been revealed to people.

[0028] In 1998, Banerjee et al. conducted a photochemical study on benzoyl methyl esters by laser flash photolysis (LFP). According to the LFP experiment, Banerjee et al. found that under 340nm ultraviolet light, benzoyl methyl esters were directly excited to a triplet state, and the triplet intermediates captured H atoms from the H-donating system (such as isopropanol) to form a semi-quinaol free radical, and isopropanol also formed a corresponding free radical. Subsequently, the semi-quinaol free radical rearranged to release the carboxylate anion, and the carrier part formed a 1-phenylvinyl alcohol intermediate, and finally rearranged to form acetophenone through the enol, and the isopropanol as the H-donating substance became acetone.

[24] (This experimental result can explain the formation of acetaldehyde and 2,3-dimethyl-2,3-diphenylbutane in the previous article).

[0029] The above experiment was conducted in a H-donating system. If it is performed in an electron-donating system, the final product is the same as that of the H-donating system. Of course, there are slight differences in the intermediates of the two systems. In the electron-donating system, the excited triplet state is still formed first, and then the O anion radical is generated, and finally the photoreaction is completed.

[25] If there is an exposed hydroxyl group at the para position of the formyl group, the product and the reaction intermediate are different. Para-hydroxybenzoyl methyl ester first generates the "Favorskii intermediate" after light exposure, and then generates the corresponding ester or carboxylic acid with the participation of ROH (R can be alkyl or H).

[26] If it is used as a photosensitive protecting group for amino acids and used in peptide synthesis, exposed hydroxyl groups are generally not allowed to exist (hydroxyl groups participate in coupling), so the protecting groups with exposed hydroxyl groups will not be explained in detail here.

[0030] Benzoylmethyl carriers are also widely used in the solid phase synthesis of peptides. In 1976, Su et al. conducted a Friedel-Crafts acylation reaction directly on the resin, introduced a bromoacetyl group on an ordinary polystyrene resin, and then coupled four amino acid residues via solid phase synthesis. Finally, a tetrapeptide was released with a yield of 70% under 350nm ultraviolet light using DMF as the solvent.

[27] .

[0031] As mentioned above, the preparation of photosensitive protective groups by modifying the resin directly is defective (it is impossible to synthesize longer peptide chains). Tjoeng and Heavner used phenylacetic acid as the starting material, introduced a bromoacetyl group at the para position of the carboxyl methyl group, and then reacted this monomer with an amino resin (carboxyl and amino groups form an amide bond) to prepare a modified phenacylmethyl photosensitive resin, and coupled five amino acid residues (with more side chain groups) through a solid phase synthesis method, and finally released a thymopentin fragment with a yield of 78%.

[28] .

[0032] Afterwards, the preparation of various types of benzoylmethyl photosensitive resins and their applications in peptide synthesis were reported one after another, which greatly enriched the research content of amino acid photosensitive carriers. Moreover, this type of carrier can also play a role in the controlled release of oligonucleotide compounds or DNA chains.

[29] According to some published literature, the suitable wavelength of benzoylmethyl photosensitive resin carriers is distributed in the range of 300-350nm, and compared with the carriers described previously, they have more active optical properties (extremely easy to be excited by light). Experimental studies on this type of carrier have shown that it has great application prospects in photochemical and biological research. At the same time, whether benzoylmethyl small molecule protecting groups can play a role in peptide synthesis is also worthy of further study.

[0033] 4. Other types of photosensitive carriers

[0034] In addition to the three common photosensitive carriers mentioned above, there are a small number of other photosensitive carriers, such as benzyl

[30] , Benzoin carrier

[31] , o-nitrobenzylamine carrier

[32] and trimethylacetyl glycol carrier

[33] (See Figure 3 , from left to right there are four types of carrier structures, the red part is the leaving group, and the black part is the parent body of the carrier).

[0035] Among them, the benzyl carrier has less nitro in its structure compared with the o-nitrobenzyl mentioned above, and its photoreaction mechanism is relatively simple. The benzyl carrier forms benzyl radicals and carboxyl radicals under light, and then forms different products according to the system.

[30] Benzoin carriers are benzoylmethyl carriers in which the H on C1 is replaced by an aromatic group. Its mechanism is similar to that of benzoylmethyl carriers, but because it has an additional benzene ring (with a larger conjugated system), the free radical intermediate formed is theoretically more stable. Therefore, it has higher optical activity than the unsubstituted benzoylmethyl carrier.

[31] The application of o-nitrobenzylamine carriers is relatively rare, and its photochemical process and mechanism are not yet clear, but it can release amine target products after light exposure.

[32] Since trimethylacetyl glycol carriers do not have a good conjugated structure, their optical activity is poorer than other carriers and generally require a shorter wavelength of light (below 300nm). Therefore, although they can release carboxylic acid under light, the parent product of the carrier is relatively complex, generally forming acetone, isobutylene and CO gas.

[34] .

[0036] References

[0037] [1]Theodor Curtius.Ueber einige neue der analogconstituirte,synthetic dargestellte Journal Für Praktische Chemie,1882,26(1),145-208.

[0038] [2] Majima, R, T. Hoshino. Synthetische Versuche in der Indol-Gruppe, VI., Eine neue Synthese von β-Indolyl-alkylaminen [J]. Berichte der deutschenchemischen Gesellschaft (A and B Series), 1925, 58 (9), 2042-2046.

[0039] [3]Bodanszky M,Tolle J C,et al.Synthesis of 8-L-tryptophan-oxytocinand determination of one of its conformational parameters[J].InternationalJournal of Peptide&Protein Research,1981,17(4),444-453.

[0040] [4]Merrifield R B.Solid phase peptide synthesis.I.The synthesis of atetrapeptide[J].Journal of the American Chemical Society,1963,85(14),2149-2154.

[0041] [5]Mikkelsen Remi J T,Grier Katja E,Mortensen Kim T,Nielsen Thomas E,Qvortrup Katrine.Photolabile Linkers for Solid-Phase Synthesis[J].ACSCombinatorial Science,2018,20,7,377-399.

[0042] [6]Rich D H,Gurwara S K.Removal of protected peptides from an ortho-nitrobenzyl resin by photolysis[J].Journal of the Chemical Society,ChemicalCommunications,1973(17),610-611.

[0043] [7]Góngora-Benítez,Miriam,Tulla-Puche J,Albericio F.Handles for FmocSolid-Phase Synthesis of Protected Peptides[J].ACS Combinatorial Science,2013,15(5),217-228.

[0044] [8]Tazhe Veetil A, Ngoy B P,et al.Photochemistry of S-Phenacyl Xanthates[J].The Journal of Organic Chemistry,2011,76,20,8232-8242.

[0045] [9]Kaja Sitkowska,Hoes M F,Lerch M M,et al.Red-light-sensitive BODIPYphotoprotecting groups for amines and their biological application incontrolling heart rhythm[J].Chemical Communications,2020,56,5480-5483.

[0046]

[10] Kulikov A,Arumugam S,Popik V V.Photolabile Protection ofAlcohols,Phenols,and Carboxylic Acids with 3-Hydroxy-2-Naphthalenemethanol[J].Journal of Organic Chemistry,2008,73(19),7611-7615.

[0047]

[11] Yu H,Li J,Wu D,et al.Chemistry and biological applications ofphotolabile organic molecules[J].Chemical Society Reviews,2010,39(2),464-473.

[0048]

[12] Yuna J,Junyang J,Youngbuhm H,et al.Benzo[g]coumarin-BasedFluorescent Probes for Bioimaging Applications[J].Journal of AnalyticalMethods in Chemistry,2018,2018,1-11.

[0049]

[13] Chen X,Wang F,Hyun J Y,et al.Recent progress in the developmentof fluorescent,luminescent and colorimetric probes for detection of reactiveoxygen and nitrogen species[J].Chemical Society Reviews,2016,45,2976-3016.

[0050]

[14] Givens R S,Matuszewski B.Photochemistry of phosphate esters,Anefficient method for the generation of electrophiles[J].Journal of theAmerican Chemical Society,1984,106(22),6860-6861.

[0051]

[15] Lin W,Lawrence D S.A strategy for the construction of caged diolsusing a photolabile protecting group[J].Journal of Organic Chemistry,2002,67(8),2723-2726.

[0052]

[16] Schade,Hagen V,Schmidt R,et al.Deactivation Behavior andExcited-State Properties of(Coumarin-4-yl)methyl Derivatives[J].The Journalof Organic Chemistry,1999,64,25,9109-9117.

[0053]

[17] Petr, et al.Photoremovable Protecting Groups inChemistry and Biology,Reaction Mechanisms and Efficacy[J].Chemical Reviews,2012,113(1),119-191.

[0054]

[18] Meng Xiangming, Chen Xiaoyun, Fu Yao, et al. Photoswitches and their applications in chemical biology[J]. Progress in Chemistry, 2008, 20(012), 2034-2044.

[0055]

[19] Barltrop JA, Plant PJ, Schofield P.Photolabile protective groups[J].Chemical Communications, 1966(22),822-823.

[0056]

[20] Il"Ichev YV,Wirz J.Rearrangements of 2-NitrobenzylCompounds.1.Potential Energy Surface of 2-Nitrotoluene and Its IsomersExplored with ab Initio and Density Functional Theory Methods[J].The Journal of Physical Chemistry A,2000,104(33),7856-7870.

[0057]

[21] Daniel H.Rich,SKGurwara.Preparation of a new o-nitrobenzylresin for solid-phase synthesis of tert-butyloxycarbonyl-protected peptideacids[J].Chemischer Informationsdienst,1975,97,1575-1579.

[0058]

[22] Klan P,Wirz J,Petr Klan,Jakob Wirz.Photochemistry of OrganicCompounds,From Concepts to Practice[M].Wiley-Blackwell,2009,ISBN,978-1-4051-6173-2.

[0059]

[23] Sheehan,J.C.,Umezawa,K.Phenacyl photolabile blocking groups[J].Journal of Organic Chemistry,1973,38(21),3771-3774.

[0060]

[24] Banerjee A,Falvey D E.Direct Photolysis of Phenacyl ProtectingGroups Studied by Laser Flash Photolysis,An Excited State Hydrogen AtomAbstraction Pathway Leads to Formation of Carboxylic Acids and Acetophenone[J].Journal of the American Chemical Society,1998,120(12),2965-2966.

[0061]

[25] Anamitro Banerjee,Daniel E.Falvey,et al.Protecting Groups ThatCan Be Removed through Photochemical Electron Transfer,Mechanistic andProduct Studies on Photosensitized Release of Carboxylates from PhenacylEsters[J].Journal of Organic Chemistry,1997,62(18),6245-6245.

[0062]

[26] Richard S.Givens,Chan-Ho Park.p-Hydroxyphenacyl ATP 1,A newphototrigger[J].Tetrahedron Letters,1996,37(35),6259-6262.

[0063]

[27] Su-Sun,Wang.Solid phase synthesis of protected peptides viaphotolytic cleavage of the alpha-methylphenacylester anchoring linkage[J].Journal of Organic Chemistry,1976,41,3258-3261.

[0064]

[28] Tjoeng,Heavner.4-(2-Chloropropionyl)phenylacetamidomethyl-resinand its application in solid-phase peptide synthesis[J].Journal of OrganicChemistry,1983,48(3),355-359.

[0065]

[29] Flickinger S T,Patel M,Binkowski B F,et al.Spatial photoreleaseof oligonucleotides,using a safety-catch photolabile linker[J].OrganicLetters,2006,8(11),2357-2360.

[0066]

[30] Zhou L,Yang H,Wang P.Development of trityl-based photolabilehydroxyl protecting groups[J].Journal of Organic Chemistry,2011,76(15),5873-5881.

[0067]

[31] Viju Balachandran Kammath,Peter et al.Photoremovable chiralauxiliary[J].Photochemical&Photobiological Sciences,2012,11,500-507.

[0068]

[32] Debieux JL, Bochet C G. Preparation of photoactivable amino acidderivatives[J]. Journal of Organic Chemistry, 2009, 74, 12, 4519-4524.

[0069]

[33] Kessler M, Glatthar R, Giese B, et al. Sequentially photocleavableprotecting groups in solid-phase synthesis [J]. Organic Letters, 2003, 5(8), 1179-1181.

[0070]

[34] Glatthar R,Giese B.Anew photocleavable linker in solid-phasechemistry for ether cleavage[J].Organic Letters,2000,2,15,2315-2317. Summary of the invention

[0071] In order to avoid the shortcomings of the prior art, the present invention provides a method for preparing a novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivatives, which mainly involve phospho (or phosphine) acyloxy coumarin methanol and its derivatives, 2-nitro-phospho (or phosphine) acyloxy benzyl alcohol and its derivatives, and α-halogenated phospho (or phosphine) acyloxy acetophenones; the present invention solves the problems that the amino acid protecting groups and solid phase resins in the current polypeptide chemical synthesis methods are easily destroyed or decomposed in the deprotection or cleavage steps and cannot be directly recycled and reused, resulting in complex reaction by-products of liquid phase synthesis, many separation steps, long time-consuming cycle, small purification scale, high production cost, and small production scale of solid phase synthesis, expensive raw material prices, large waste, a large amount of resin waste and serious environmental pollution.

[0072] To achieve the above purpose, the technical solution provided by the present invention is:

[0073] The novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivatives are special in that:

[0074] The carboxylic acid protecting group that can be photocatalytically cleaved is a 2-nitro-phosphoryloxybenzyl compound 1, and its amino acid derivative

[0075] The general molecular structure of organism 2 is:

[0076]

[0077] Alternatively, the general molecular structure formula of the phosphoryloxy-4-coumarinylmethyl compound 3 and its amino acid derivative 4 is:

[0078]

[0079] Alternatively, the molecular structure formula of the phosphoryloxyphenacyl compound 5 and its amino acid derivative 6 is:

[0080]

[0081] Wherein, R represents the side chain residue of various amino acids.

[0082] The nitro group in compound 1 above facilitates photocleavage, while compounds 3 and 5 can be directly photocleaved.

[0083] At the same time, the present invention also provides a method for preparing the above-mentioned novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivatives, which is special in that it comprises the following steps:

[0084] 1) Using (R)2POCl or POCl3 and coumarin methyl halide, 2-nitrobenzyl halide or α-haloacetophenone containing a hydroxyl group on the benzene ring as raw materials, reacting in an alkaline solution, and separating and purifying to obtain the corresponding phosphoryloxy derivative;

[0085] 2) using the phosphoryloxy derivative obtained in step 1) as a raw material, hydrolyzing or converting the corresponding halogen functional group, and obtaining the corresponding derivatized product, a phosphorus-substituted methanol compound, through separation and purification;

[0086] 3) condensing and coupling the phosphorus-containing substituted methanol compound obtained in step 2) with an Fmoc-protected amino acid (Fmoc-AA-OH) or a Boc-protected amino acid (Boc-AA-OH), and obtaining the corresponding Fmoc-amino acid ester derivative product or Boc-amino acid ester derivative product of the compound through separation and purification.

[0087] Furthermore, the novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivative are 2-nitro-phosphoryloxybenzyl compound 1 and its amino acid derivative 2, comprising the following steps:

[0088] Step 1: using vanillic aldehyde or vanillin ketone as a raw material to react with (R)2POCl or POCl3 respectively, adding a base to a solvent A to catalyze the reaction, after the reaction is completed, recovering the solvent by rotary evaporation, extracting the residual solution with ethyl acetate and separating the aqueous phase, combining the organic phases and washing them with deionized water for multiple times; separating and purifying to obtain a phosphoryloxy vanillic aldehyde compound or a phosphoryloxy vanillin ketone compound;

[0089] Then, the obtained phosphoryloxy vanillic aldehyde compound or phosphoryloxy vanillic ketone compound is nitrated with nitric acid, and separated and purified to obtain 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic aldehyde ketone compound;

[0090] Step 2: using the 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic aldehyde ketone compound obtained in step 1 as a raw material, and using sodium borohydride as a reducing agent to carry out a reduction treatment reaction in a solvent B. After the reaction is completed, concentrated hydrochloric acid is added to adjust the pH value to 2-3 to terminate the reaction, and the solvent is recovered by rotary evaporation. The residual solution is extracted with ethyl acetate and separated from the aqueous phase, and the organic phases are combined and washed with deionized water for multiple times; then separation and purification are performed to obtain the corresponding derivative product 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic ketone compound, 2-nitro-phosphoryloxy benzyl alcohol compound;

[0091] A 2-nitro-phosphoryloxybenzyl alcohol compound is used as a raw material, reacted with concentrated hydrohalic acid under the catalysis of anhydrous zinc halide, after the reaction is completed, the solvent is recovered by rotary evaporation, the residual solution is extracted with ethyl acetate and separated from the aqueous phase, the organic phases are combined and washed with deionized water for multiple times; then, the corresponding derivative product of the 2-nitro-phosphoryloxybenzyl compound, i.e., a 2-nitro-phosphoryloxybenzyl halogen compound, is separated and purified;

[0092] The 2-nitro-phosphoryloxybenzyl halogen compound is reacted with sodium amide or sodium hydrosulfide to be converted into the corresponding 2-nitro-phosphoryloxybenzylamine compound or 2-nitro-phosphoryloxybenzylthiol compound, the general structural formula of which is shown in Compound 1, and they are collectively referred to as 2-nitro-phosphoryloxybenzyl compounds;

[0093] Step 3: The 2-nitro-phosphoryloxybenzyl alcohol compounds, 2-nitro-phosphoryloxybenzyl halogen compounds, 2-nitro-phosphoryloxybenzyl amine compounds or 2-nitro-phosphoryloxybenzyl thiol compounds obtained in step 2 are respectively subjected to coupling reaction with Boc- or Fmoc-protected amino acids (PG-AA-OH), and their corresponding amino acid derivatives 2 are obtained after separation and purification.

[0094] Alternatively, further, the novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivative are phosphoryloxy-4-coumarinylmethyl compound 3 and its amino acid derivative 4, comprising the following steps:

[0095] 1) using hydroxycoumarin-4-methyl halogen as a raw material to react with (R)2POCl or POCl3 respectively, adding a base to a solvent A to catalyze the reaction, after the reaction is completed, recovering the solvent by rotary evaporation, extracting the residual solution with ethyl acetate and separating it from the aqueous phase, combining the organic phases and washing them with deionized water for multiple times, separating and purifying to obtain a phosphoryloxycoumarin-4-methyl halogen compound;

[0096] Using a phosphoryloxycoumarin-4-methyl halogen compound as a raw material, reacting with sodium hydroxide, sodium amide or sodium hydrosulfide, respectively, to convert into a corresponding phosphoryloxycoumarin-4-methanol compound, a phosphoryloxycoumarin-4-methylamine compound or a phosphoryloxycoumarin-4-methylthiol compound, whose chemical structure is shown in general formula 3, collectively referred to as phosphoryloxy-4-coumarinyl methyl compounds;

[0097] 2) The phosphoryloxycoumarin-4-methyl halogen compounds, phosphoryloxycoumarin-4-methanol compounds, phosphoryloxycoumarin-4-methylamine compounds or phosphoryloxycoumarin-4-methylthiol compounds obtained in step 1) are respectively subjected to coupling reaction with Boc- or Fmoc-protected amino acids (PG-AA-OH), and their corresponding amino acid derivatives 4 are obtained after separation and purification.

[0098] Alternatively, further, the novel photocatalytically cleavable carboxylic acid protecting group and its amino acid derivative are phosphoryloxyphenacyl compound 5 and its amino acid derivative 6, comprising the following steps:

[0099] S1. Using paeonol or its substituted derivatives as raw materials, respectively, reacting with (R)2POCl or POCl3, adding a base to a solvent A for catalysis, and after the reaction is completed, recovering the solvent by rotary evaporation, extracting the residual solution with ethyl acetate and separating the aqueous phase, combining the organic phases and washing them with deionized water for multiple times, separating and purifying to obtain 2-phosphoryloxyacetophenone compounds;

[0100] S2. Using the phosphoryloxyacetophenone compound or its substitution product obtained in S1 as a raw material, adding copper bromide to the C solvent and stirring the reaction, after the reaction is completed, the solvent is recovered by rotary evaporation, the residual solution is extracted with ethyl acetate and separated from the aqueous phase, the organic phases are combined and washed with deionized water for multiple times, and the phosphoryloxyphenacyl bromide is separated and purified;

[0101] The phosphobenzoyl bromide is treated with a sodium hydroxide aqueous solution, an alcohol solution of sodium amide or an alcohol solution of sodium hydrosulfide, respectively, to separate and purify to obtain phosphobenzoylmethanol compounds, phosphobenzoylamine compounds or phosphobenzoylthiol compounds, whose chemical structures are shown in general formula 5, collectively referred to as phosphobenzoyl compounds;

[0102] S3. The phosphooxyphenacyl bromide, phosphooxyphenacylmethanol compounds, phosphooxyphenacylamine compounds and phosphooxyphenacylthiol compounds obtained in S2 are respectively subjected to coupling reaction with Boc- or Fmoc-protected amino acids (PG-AA-OH), and their corresponding amino acid derivatives 6 are obtained after separation and purification.

[0103] Furthermore, the feed ratio of (R)2POCl or POCl3 to vanillic aldehyde or vanillic ketone is 1 to 4:1, the reaction temperature is -5 to 50°C, and the reaction time is 1 to 5 hours;

[0104] The feeding ratio of the nitric acid to the phosphoryloxy vanillic aldehyde compound or the phosphoryloxy vanillic ketone compound is 1:1-3, the reaction temperature is -5-50°C, and the reaction time is 1-5 hours.

[0105] Furthermore, the feed ratio of (R)2POCl or POCl3 to hydroxycoumarin-4-methyl halogen is 1 to 4:1, the reaction temperature is -5 to 50°C, and the reaction time is 1 to 5 hours.

[0106] Furthermore, the feed ratio of (R)2POCl or POCl3 to paeonol or its substituted derivative is 1 to 4:1, the reaction temperature is -5 to 50°C, and the reaction time is 1 to 5 hours;

[0107] The feeding ratio of the phosphoryloxyacetophenone compound to copper bromide is 1:1-4, the reaction temperature is -5-50°C, and the reaction time is 5-12 hours;

[0108] The feeding ratio of the sodium hydroxide, sodium amide or sodium hydrosulfide to phosphoryloxyphenacyl bromide is 1:1-3, the reaction temperature is -5-50°C, and the reaction time is 1-5 hours.

[0109] Furthermore, the separation and purification is: extracting with ethyl acetate and separating with water phase, combining organic phases and washing with deionized water 2-3 times; adding a small polar alkane or ether solvent to the ethyl acetate solution until crystals or precipitations are formed, and completing the separation and purification by filtering and washing or recrystallization;

[0110] The solvent A is one or more of tetrahydrofuran, acetonitrile, benzene, toluene, chloroform, dichloromethane, N,N-dimethylformamide, and N-methylpyrrolidone;

[0111] The base is one or more of tertiary amines TEA, DIEA, NMM, pyridine and DMAP.

[0112] Furthermore, the reaction conditions of the base-adding catalytic reaction and the sodium borohydride reduction reaction are: stirring at room temperature for 2 to 3 hours;

[0113] The number of times of washing with deionized water is 2 to 3 times;

[0114] The B solvent is one or more of alcohols, acetonitrile, or tetrahydrofuran;

[0115] The C solvent is one or more of ethyl acetate, acetonitrile, tetrahydrofuran, chloroform, dichloromethane, or DMF;

[0116] The alkane or ether solvent is one or more of petroleum ether, n-hexane, cyclohexane, diethyl ether, or n-butyl ether.

[0117] The advantages of the present invention are:

[0118] 1. The raw materials used for the synthesis of the protecting group of the present invention are easily available, the synthesis steps are simple, and the organic phosphorus group has the property of assisting precipitation, which is beneficial to the separation and purification of the intermediate and the target product.

[0119] 2. The protecting groups are cleaved under photocatalysis under mild conditions, and the cleaved protecting groups are easy to recycle and reuse, which not only avoids excessive use of acid and alkali, but also reduces the discharge of three wastes, is green and environmentally friendly, and saves costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Figure 1 It is a photoshearing reaction;

[0121] Figure 2 The first application of photosensitive resin [6] ;

[0122] Figure 3 For the other four photosensitive carrier structures;

[0123] Figure 4 HPLC spectra of standard and reaction solution;

[0124] Figure 5 is the high-resolution mass spectrum of the reaction solution;

[0125] Figure 6 HPLC graphs of photo-shearing reaction in acetonitrile at different time periods;

[0126] Figure 7 This is the kinetic curve of photoshearing reaction. DETAILED DESCRIPTION

[0127] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0128] A method for preparing a novel carboxylic acid protecting group capable of photocatalytic cleavage and its amino acid derivatives, characterized in that the molecular structure of the carboxylic acid protecting group compound is as follows:

[0129]

[0130]

[0131] Phosphonylation of vanillin

[0132] 5-methoxy-4-(diphenylphosphinoyloxy)benzaldehyde (1) was prepared by reacting vanillin with diphenylphosphinoyl chloride. 5.07 g (33 mmol) of vanillin was accurately weighed and placed in a 250 mL round-bottom flask, and 80 mL of dichloromethane was added to dissolve it. 3.45 g (34 mmol, about 4.7 mL) of triethylamine was added in a fume hood, and the mixture was sealed and placed in an ice-water bath and stirred for 10 min to allow the entire system to cool down sufficiently. 8.04 g (34 mmol, about 6.5 mL) of diphenylphosphinoyl chloride was slowly added dropwise to the flask in 4 portions, and stirred in an ice-water bath to mix evenly. During the addition of diphenylphosphinoyl chloride, the system gradually became turbid, and a large amount of white smoke was generated at the mouth of the flask (hydrogen chloride and triethylamine were generated by the reaction). Amine reacts to form white smoke); TLC spot plate monitors the reaction (developing agent EA:PE=1:1, V:V). After 10 minutes of adding diphenylphosphinoyl chloride, vanillin is completely consumed. 5 mL of saturated ammonium chloride aqueous solution is added and stirred to quench the reaction. After stopping the reaction, the organic phase is washed twice with 80 mL of saturated NaHCO3 aqueous solution and saturated brine, dried over anhydrous MgSO4, filtered, and after rotary evaporation to remove dichloromethane, 15 mL of cold methanol is used for slurrying (to remove a small amount of residual triethylamine hydrochloride). After filtering and drying, 10.1 g of white powder is obtained, and the yield is 87%. Product structure characterization 5-methoxy-4-(diphenylphosphinoyloxy)benzaldehyde (C 20 H 17 O4P): 1 H NMR (400MHz, DMSO-d6) δ9.87 (s, 1H), 7.90 (ddd, J = 12.5, 8.2, 1.5Hz, 4H), 7.63–7.47 (m, 9H), 3.89 (s, 3H); 13 C NMR(100MHz,DMSO-d6)δ192.17,151.39,144.93,134.00,133.40,131.87, 131.77,131.62,130.26,129.46,129.33,124.36,121.90,112.46,56.54; 31P NMR(162MHz,DMSO-d6)δ31.06; HRMS(ESI)m / z calcd forC 20 H 17 O4PNa + (M+Na) + 375.07567, found 375.07599.

[0133] Nitration of Vanillin Phosphate

[0134] There is a methoxy group in the 5-methoxy-4-(diphenylphosphinoyloxy)benzaldehyde molecule, which is an ortho-para-positioning group and can introduce a nitro group at the target position. Four nitration systems were tried: acetic anhydride-potassium nitrate, acetic anhydride-copper nitrate, acetic anhydride-nitric acid and trifluoroacetic acid-nitric acid. The nitration rates of acetic anhydride-potassium nitrate and acetic anhydride-nitric acid were too slow, especially the acetic anhydride-potassium nitrate system, which still had a large part of unreacted after 12 hours of reaction (TLC spot plate to see the spot concentration), while the trifluoroacetic acid-nitric acid system had too many side reactions. Finally, it was found that the nitration effect of the acetic anhydride-copper nitrate system was more satisfactory, and the TLC plates of the four systems (developing solvent was DCM:MeOH=15:1) were shown. Accurately weigh 7.04 g (20 mmol) of 5-methoxy-4-(diphenylphosphinoyloxy)benzaldehyde (Compound 1), place it in a 250 mL round-bottom flask, add 50 mL of acetic anhydride to fully dissolve it, place the flask in a 40°C oil bath to preheat for 10 min, then add 7.25 g (30 mmol) of copper nitrate trihydrate, continue heating at 40°C for 20 min, and a light yellow color can be observed in the solution. Then move the flask to an ice water bath to continue the reaction; monitor the reaction by TLC spot plate (developing agent D CM:MeOH=15:1,V:V), moved to ice water bath for 2h, 5-methoxy-4-(diphenylphosphinoyloxy)benzaldehyde was completely consumed, the reaction solution was poured into 300mL cold water, and stirring was continued for 1h. During the stirring process, a large number of light yellow oily droplets were generated. After standing, the droplets gathered and adhered to the bottom of the beaker and turned into solid. After skimming the supernatant, it was washed twice with 50mL deionized water, and then recrystallized with 30mL methanol. After washing and drying, 5.1g of beige large granular crystals were obtained, and the yield was 64%. Product structure characterization 5-methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzaldehyde (2, C 20 H 16 NO6P): 1 H NMR(400MHz,DMSO-d6)δ8.14(s,1H),8.10(s,1H),7.94–7.88(m,4H),7.68(t d,J=7.4,1.5Hz,2H),7.60(dt,J=7.4,3.7Hz,4H),7.35(s,1H),4.00(s,3H);13 C NMR(100MHz,DMSO-d6)δ168.91,155.72,139.98,139.18,133.70,131.89,131 .78,131.09,129.74,129.60,129.46,129.39,119.07,111.70,85.16,57.51; 31 P NMR(162MHz,DMSO-d6)δ33.12; HRMS(ESI)m / zcalcd for C 20 H 16 NO6PNa + (M+Na) + 420.06074, found 420.06085.

[0135] Reduction of 5-methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzaldehyde

[0136] 5-Methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzaldehyde was reduced using NaBH4. Sodium borohydride is a mild and chemically selective reducing agent that can rapidly reduce aldehydes and ketones to primary and secondary alcohols in solvents containing hydroxyl groups (such as water and alcohols) at room temperature. Accurately weigh 3.97 g (10 mmol) of 5-methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzaldehyde, place it in a 250 mL round-bottom flask, add 40 mL of methanol and 60 mL of dichloromethane respectively to fully dissolve it (sodium borohydride has good reducing properties in solvents containing hydroxyl groups, simple methanol has poor solubility, and dichloromethane acts as a solubilizing agent). Then place the round-bottom flask in an ice-water bath and stir for 10 min to allow the entire system to cool down sufficiently. Subsequently, 0.756 g (20 mmol) of sodium borohydride is added in batches in 8 times, and stirring is continued in the ice-water bath. As the NaBH4 solid gradually dissolves, the solution gradually turns orange-yellow, and a large number of dense bubbles appear (the flask needs a bottle stopper with a balloon to keep it sealed and ensure safety). The bubbles are hydrogen generated by the reaction of NaBH4 and alcohol. In the absence of a metal catalyst, elemental hydrogen has no reducing properties to carbonyl groups, so Na BH4 needs to be added in excess and in batches; 30 minutes after the last batch of sodium borohydride is added, the bubbles in the system disappear, NaBH4 is completely consumed, and the reaction is monitored by TLC spot plate (developing agent DCM:MeOH=15:1, V:V). 5-methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzaldehyde reacts completely, and stirring is continued in an ice-water bath. 1 mol / L dilute hydrochloric acid solution (to neutralize the alkali produced) is slowly added dropwise to adjust the pH value to 7-8. With the addition of hydrochloric acid, a large amount of light yellow solid precipitates, and the orange color of the solution gradually fades. After adjusting the pH value, the flask is moved to a rotary evaporator to remove methanol and dichloromethane in the system, and then the aqueous phase is extracted 3 times with dichloromethane. After the organic phases are combined, they are washed twice with saturated brine, dried over anhydrous MgSO4, and then rotary evaporated to remove dichloromethane. Finally, 3.1 g of beige needle-shaped crystals (ONBG) are obtained by recrystallization with anhydrous ethanol, with a yield of 77%. Product structure characterization 5-methoxy-4-(diphenylphosphinoyloxy)-2-nitrobenzyl alcohol (3, ONBG, C 20 H 18 NO6P): 1 H NMR(400MHz, DMSO-d6)δ8.09(s,1H),7.95–7.84(m,4H),7.71–7.63(m,2H),7.58(td,J= 7.5, 3.6Hz, 4H), 7.48 (s, 1H), 5.66 (t, J = 5.4Hz, 1H), 4.82 (d, J = 5.4Hz, 2H), 3.94 (s, 3H); 13C NMR(100MHz,DMSO-d6)δ155.55,139.45,138.11,133.57,133.54,131.91,131 .80,131.26,129.91,129.51,129.38,118.34,118.30,111.42,60.56,57.06; 31 P NMR(162MHz,DMSO-d6)δ32.23; HRMS(ESI)m / z calcd for C 20 H 18 NO6PNa + (M+Na) + 422.07639, found 422.07645.

[0137] Coupling reaction with Boc- or Fmoc-amino acids

[0138] Synthesis of Boc-His(Trt)-ONBG (4a): Boc-His(Trt)-OH (0.6 g, 1.2 mmol, 1.2 eq), EDC·HCl (0.3 g, 1.5 mmol, 1.5 eq) and DMAP (0.012 g, 0.1 mmol, 0.1 eq) were weighed and placed in a 50 mL round-bottom flask, and then 20 mL of dichloromethane was added to fully dissolve, and stirred in an ice-water bath for 30 min to activate (to form active ester), and then ONBG (0.4 g, 1 mmol, 1 eq) was accurately weighed. ) was added to a round-bottom flask and stirred in an ice-water bath. The system gradually turned orange. The reaction was monitored by TLC spot plate (developing solvent DCM: MeOH = 15:1, Vol). After 30 minutes, the ONBG protective group was completely reacted. The reaction was stopped and the solvent was directly removed by rotary evaporation. After the dichloromethane was dried, 5 mL of acetonitrile was added. After heating and ultrasonication, the solid was completely dissolved. After standing in a refrigerator for 10 minutes, a large amount of off-white solid precipitated. The filter cake was filtered and washed with 2 mL of ice acetonitrile. After drying, 0.76 g of powdery off-white solid was obtained with a yield of 87%. Product structure characterization Boc-His(Trt)-ONBG(C 50 H 47 N4O9P): 1H NMR (400MHz, DMSO-d6) δ8.13(s,1H),7.92–7.83(m,4H),7.66(td,J=7.4,1.5Hz,2H),7.57(td,J=7.4,3.4Hz,4H),7.36(dd,J=5.2,1.9Hz,9H ),7.30–7.26(m,2H),7.06–7.02(m,6H),6.71(s,1H),5.47–5.24(m,2H ),4.36(q,J=7.1Hz,1H),3.95(s,3H),2.93–2.79(m,2H),1.32(s,9H); 31 P NMR(162MHz,Chloroform-d)δ33.20; HRMS(ESI)m / zcalcd for C 50 H 48 N4O9P + (M+H) + 879.31534, found 879.31641.

[0139] Synthesis of Fmoc-His(Trt)-ONBG(4b): The reagent dosage, feeding sequence, reaction phenomenon and time, and post-treatment process of ONBG coupling with Fmoc-histidine Boc-His(Trt)-OH are the same as those of Boc-histidine. Finally, 0.9 g of beige solid was obtained after recrystallization from acetonitrile, with a yield of 90%. Product structure characterization Fmoc-His(Trt)-ONBG(C 60 H 49 N4O9P): 1 H NMR(400MHz, DMSO-d6)δ8.12(d,J=1.4Hz,1H),7.91–7.85(m,7H),7.68–7.61(m,4H),7.57( ddd,J=7.4,5.7,3.6Hz,4H),7.39(t,J=7.5Hz,2H),7.33(dd,J=5.1,1.9Hz,10H),7.29–7.2 2(m,4H),7.05–7.01(m,7H),6.73(d,J=1.4Hz,1H),5.55–5.26(m,2H),4.45(td,J=7.9,5.9 Hz,1H),4.27(dt,J=6.7,3.9Hz,2H),4.16(t,J=6.9Hz,1H),3.86(s,3H),2.98–2.80(m,2H); 31 P NMR (162MHz, DMSO-d6) δ32.50; HRMS (ESI) m / z calcdfor C 60 H49 N4O9PNa + (M+Na) + 1023.31294, found 1023.31329.

[0140] Synthesis Scheme 2

[0141]

[0142] Synthesis of 7-Hydroxy-4-chloromethylcoumarin (8)

[0143] Measure 100 mL of concentrated sulfuric acid and place it in a 250 mL round-bottom flask, seal it and place it in an ice-water bath and stir for 10 min to fully cool the whole system; weigh 11.01 g (100 mmol) of resorcinol and 19.75 g (120 mmol, about 16.2 mL) of ethyl 4-chloroacetoacetate and place them in a 50 mL centrifuge tube, and gently heat to dissolve resorcinol in ethyl 4-chloroacetoacetate as much as possible (do not use ultrasound, and do not heat too much, otherwise resorcinol cannot be completely dissolved and will not affect subsequent reactions); use a dropper to add isocyanate The mixture of diphenol and ethyl 4-chloroacetoacetate (the solution was mixed with a small amount of solid) was slowly added dropwise to the cooled concentrated sulfuric acid, and an ice-water bath was maintained. During the addition, the system gradually turned brown-red. After the addition was completed, the ice-water bath was removed; TLC spot plate was used to monitor the reaction (developing agent EA:PE = 1:1, Vol). After 12 hours, the reaction of resorcinol was complete. The reaction solution was slowly poured into 500 mL of ice-water mixture (a large amount of light yellow colloid was precipitated). After the pouring was completed, it was stirred at room temperature for 30 minutes. After filtering, washing, and drying, the crude product was recrystallized with 50 mL of 75% alcohol to finally obtain 15.1 g of off-white needle-shaped crystals with a yield of 72%. Product structure characterization 7-Hydroxy-4-chloromethylcoumarin (compound 8, C 10 H7ClO3): 1 H NMR (400MHz, DMSO-d6) δ10.66(s,1H),7.68(d,J=8.7Hz,1H),6.85(dd,J=8.6,2.4Hz,1H),6.76(s,1H),6.42(s,1H),4.96(s,2H); 13 C NMR (100MHz, DMSO-d6) δ161.92,160.60,155.76,151.45,127.02,113.54,111.53,109.81,102.98,41.83.

[0144] Synthesis of 7-Hydroxy-4-hydroxymethylcoumarin (9)

[0145] Accurately weigh compound 8 (10.5 g, 50 mmol), place in a 250 mL round-bottom flask, add about 30 mL DMF and 45 mL deionized water (the solid cannot be completely dissolved at room temperature, the system is a milky white suspension), heat and stir at 110 ° C, condense and reflux, react overnight; TLC spot plate monitors the reaction (developing solvent DCM: MeOH = 10: 1, Vol), after reacting overnight, compound 5 is completely consumed, the reaction solution is poured into 200 mL saturated brine, the aqueous phase is extracted three times with 200 mL ethyl acetate, the organic phase is combined, the organic phase is washed three times with saturated brine, dried over anhydrous MgSO4, filtered and evaporated to remove ethyl acetate, and an orange-yellow crude product is obtained. The crude product is recrystallized with 30 mL methanol and dried to obtain 7.392 g of a white powdery solid with a yield of 77%. Product structure characterization 7-Hydroxy-4-hydroxymethylcoumarin (compound 9, C 10 H8O4): 1 H NMR (400MHz, DMSO-d6) δ10.52(s,1H),7.52(d,J=8.7Hz,1H),6.77(dd,J=8.7,2.4Hz,1H), 6.73(d,J=2.3Hz,1H), 6.24(d,J=1.5Hz,1H), 5.59(t,J=5.6Hz,1H), 4.70(d,J=3.6Hz,2H); 13 C NMR(100MHz,DMSO-d6)δ161.44,161.09,157.27,155.31,125.93,113.28,110.00,106.98,102.75,59.53; HRMS(ESI)m / z calcd for C 10 H8O4Na + (M+Na) + 215.03148, found 215.03163.

[0146] Synthesis of 7-diphenylphosphonyloxy-4-chloromethylcoumarin (10)

[0147] Compound 9 (3.5 g, 16.67 mmol) was weighed and placed in a 100 mL round-bottom flask, 30 mL of tetrahydrofuran was added to dissolve, the mixture was sealed and placed in an ice-water bath and stirred for 10 min to allow the entire system to cool down sufficiently; 4.02 g (17 mmol, about 3.24 mL) of diphenylphosphine chloride was added to the flask, stirred to mix evenly, and then 1.72 g (17 mmol, about 2.36 mL) of triethylamine was slowly added dropwise. During the addition of triethylamine, the system gradually became turbid, and a large amount of white smoke was generated at the mouth of the flask (according to the reaction principle, triethylamine should be added first to convert the phenolic hydroxyl group in compound 5 into an oxygen anion, which is convenient for reacting with the acyl chloride, but due to the presence of triethylamine in the molecule The benzyl chloride group is very easy to react with triethylamine to form a quaternary ammonium salt, and the target product cannot be obtained); TLC spot plate monitoring reaction (developing agent DCM: MeOH = 10: 1, Vol), after half an hour, compound 5 is completely consumed, 5 mL of saturated ammonium chloride aqueous solution is added to quench the reaction, tetrahydrofuran is removed by rotary evaporation, 50 mL of dichloromethane is added to dissolve the solid, the organic phase is washed three times with 50 mL of saturated brine, dried and filtered, and after rotary evaporation to remove dichloromethane, 4.75 g of beige solid is obtained by column chromatography (eluent DCM: MeOH = 30: 1, Vol), and the yield is 69%. Although the triethylamine is controlled to be added last, the final yield of this step is still not very high. Product structure characterization 7-diphenylphosphonyloxy-4-chloromethyl coumarin (compound 10, CPPG, Couamrin-Bsaed PhotolabileProtecting Group, C 22 H 16 ClO4P): 1H NMR (400MHz, DMSO-d6) δ8.03–7.94(m,4H),7.82(d,J=8.7Hz,1H),7.62(ddd,J=23.8,7.5,2.7Hz,6H),7.45–7.33(m,2H),6.61(s,1H),4.98(s,2H); 13 C NMR(100MHz,DMSO-d6)δ159.80,154.56,153.86,150.65,133.58,133.55,132.07,131.97, 131.24,129.88,129.61,129.48,127.34,117.58,114.87,114.53,109.28,109.22,41.65; 31 P NMR(162MHz,DMSO-d6)δ30.95; HRMS(ESI)m / z calcd for C 22 H 17 ClO4P + (M+H) +411.05475, found 411.05490.

[0148] Synthesis of Coumarin Protective Group and Amino Acid Coupling Derivatives (11)

[0149] The coupling effects of these coumarin-based photosensitive protecting groups with amino acids were explored experimentally to verify whether they can be used as carboxyl-terminal protecting groups of amino acids, and then the photo-shearing properties of compounds coupled with coumarin-based photosensitive protecting groups and single amino acids were studied. At the same time, in order to verify whether peptide synthesis can be carried out after coupling the protecting groups with amino acids, the Boc / Fmoc group of the coupled amino acids needs to be removed. The four designed and synthesized coumarin-based photosensitive protecting groups can be divided into two categories according to the active site: benzyl chloride and benzyl alcohol. Among them, the esterification coupling reaction of the benzyl alcohol protecting group with Fmoc- or Boc-amino acids can use traditional coupling reagents EDC·HCl / DMAP or DCC / DMAP, and the reaction is easy to carry out and the yield is high. However, benzyl chloride cannot directly undergo esterification reaction, and it is necessary to convert the carboxyl group of the amino acid into a carboxyl anion under alkaline conditions (potassium fluoride), and then undergo nucleophilic substitution reaction to form esters.

[0150] Boc-Val-OH or Boc-Phe-OH (1.1 mmol, 1.1 eq), EDC·HCl (0.288 g, 1.5 mmol, 1.5 eq), and DMAP (12.5 mg, 0.1 mmol, 0.1 eq) were weighed and placed in a 100 mL round-bottom flask, 30 mL of dichloromethane was added, and the mixture was stirred in an ice-water bath for 30 min to fully activate the system (to generate active esters), and then compound 10 (0.392 g, 1 mmol, 1 eq) was weighed and added to the reaction solution, and stirring was continued at room temperature. The reaction was monitored by TLC spot plate (developing solvent DCM: MeOH = 10:1, Vol). After one hour, compound 10 was completely reacted. The reaction solution was washed twice with 30 mL of saturated NH4Cl aqueous solution, saturated NaHCO3 aqueous solution and saturated brine, dried over anhydrous MgSO4 and then dichloromethane was removed by rotary evaporation. 2 mL of ethyl acetate was added to dissolve the sample again. 15 mL of petroleum ether was slowly dropped into the solution. The oscillation / ultrasound was maintained. A colloidal precipitate appeared in the system and adhered to the wall of the bottle. After standing, the clear liquid was poured out and 10 mL of ethyl acetate and petroleum ether were mixed. The precipitate was washed with solvent (volume ratio 1:8), and the supernatant was skimmed off and the residual solvent was removed by rotary evaporation and vacuum drying to obtain 0.605 g of a white foamy solid (Boc-Phe-CPPG, a coupling product of CPPG and Boc-Phe-OH) with a yield of 95%, and 0.51 g of a light yellow foamy solid (Boc-Val-CPPG, a coupling product of CPPG and Boc-Val-OH) with a yield of 86%. (In the following, for ease of expression, compound 10 is abbreviated as CPPG, Couamrin-Bsaed Photolabile Protecting Group).

[0151] Boc-Phe-CPPG(11a,C 36 H 34 NO8P): 1 H NMR (400MHz, DMSO-d6) δ7.98–7.95(m,4H),7.67–7.38(m,10H),7.26–7.16(m,5H),6.38(s,1H),5.34( d,J=6.7Hz,2H),4.04(t,J=7.1Hz,1H),3.03(dd,J=13.7,5.6Hz,1H),2.99–2.90(m,1H),1.32(s,9H); 13C NMR(100MHz,DMSO-d6)δ172.06,159.67,155.98,154.27,149.91,137.76,133.55,133.52,132.07,131.97,131.26,129.90,129.59,129.55,129.45,128.65,126.95,126.80,117.51,114.19,111.66,109.14,78.99,61.96,55.88,36.70,28.53; 31 P NMR(162MHz,DMSO-d6)δ30.87;HRMS(ESI)m / z calcd for C 36 H 34 NO8PNa + (M+Na) + 662.19142,found 662.19147.

[0152] Boc-Val-CPPG(11b,C 32 H 34 NO8P): 1 H NMR(400MHz,DMSO-d6)δ8.00–7.93(m,4H),7.71(d,J=8.8Hz,1H),7.68–7.62(m,2H),7.58(dt,J=8.9,3.6Hz,4H),7.48–7.40(m,2H),6.47(d,J=1.6Hz,1H),5.51–5.30(m,2H),3.94(t,J=7.3Hz,1H),2.07–1.98(m,1H),1.38(s,9H),0.89(dd,J=6.8,4.2Hz,7H); 13 C NMR(100MHz,DMSO-d6)δ172.02,159.71,156.40,154.26,150.19,133.56,133.53,132.07,131.97,131.23,129.87,129.59,129.46,126.80,117.50,114.20,111.57,109.16,109.11,78.92,61.79,60.22,29.86,28.61,28.30,19.46,18.93;HRMS(ESI)m / z calcd for C 32 H 35 NO8P + (M+H) + 592.20948,found 592.20990.

[0153] The coumarin-based protecting group of benzyl alcohol can be esterified with amino acids by using the traditional coupling reagent EDC·HCl / DMAP, and after the coupling is completed, the product can be purified by a method similar to solid phase precipitation (chromatography can be avoided), and the yield of coupling with amino acids is also satisfactory. Therefore, the coumarin-based protecting group of benzyl alcohol can be directly used as the carboxyl terminal protection of amino acids.

[0154] Synthesis of tris(4-chloromethyl-7-coumarinyl) phosphate (12)

[0155] Compound 8 (3.5 g, 16.67 mmol) was weighed and placed in a 100 mL round-bottom flask. 30 mL of tetrahydrofuran was added to dissolve the mixture. The mixture was sealed and placed in an ice-water bath and stirred for 10 min to allow the entire system to cool down sufficiently. 1.72 g (17 mmol, about 2.36 mL) of triethylamine was added to the flask. 0.85 g (5.56 mmol, about 0.52 mL) of phosphorus oxychloride was slowly added dropwise in three batches. The mixture was stirred in an ice-water bath. During the addition of phosphorus oxychloride, the system gradually became turbid. A large amount of white smoke was generated; the reaction was monitored by TLC spot plate (developing solvent DCM: MeOH = 10:1, Vol). After half an hour, compound 5 was completely consumed. 5 mL of saturated ammonium chloride aqueous solution was slowly added to quench the reaction. THF was removed by rotary evaporation. 10 mL of methanol was added. A large amount of white solid did not dissolve (compound 11 had poor solubility in methanol). Ultrasound was used to fully dissolve the impurities. After filtering and washing, 1.34 g of white powdery solid was obtained. The yield was 35% (most of the raw materials formed quaternary ammonium salts with triethylamine). Product structure characterization Tris (4-chloromethyl-7-coumarinyl) phosphate (compound 12, TCCP, C 30 H 18 Cl3O 10 P): 1 H NMR (400MHz, DMSO-d6) δ7.96(d,J=8.8Hz,3H),7.55(d,J=2.5Hz,3H),7.47(dd,J=8.8,2.5Hz,3H),6.71(s,3H),5.04(s,6H); 13 C NMR (100MHz, DMSO-d6) δ159.68,154.75,152.07,150.45,127.85,116.92,115.85,115.72,109.28,41.66; 31 P NMR(162MHz,DMSO-d6)δ-18.99; HRMS(ESI)m / z calcd for C 30 H 19 Cl3O 10 P+ (M+H) + 674.97759, found 674.97742.

[0156] Tris(4-chloromethyl-7-coumarinyl)phosphate (Compound 12, TCCP) cannot undergo esterification reaction directly. It needs to be reacted under alkaline conditions (potassium fluoride) to convert the carboxyl group of the amino acid into a carboxyl anion, and then undergo a nucleophilic substitution reaction to generate amino acid esters (see the general formula of Compound 13).

[0157] Photoshear reaction test

[0158] The photo-shearing mechanism of coumarin has been studied thoroughly. Schade et al. 18 O photocleaves the coumarin-phosphate ester, releasing free phosphate and coumarinylmethanol. 18 O participates in the formation of coumarinylmethanol.

[0159] The photo-shearing study of CPPG protecting group was carried out systematically through the synthesized Boc-Phe-CPPG compound, and the reaction results and process were studied by high performance liquid chromatography (HPLC). The hydrolysis of Boc-Phe-CPPG compound under light needs to be carried out in water and aprotic solvent (polar aprotic solvent can ensure that the solvent provides H and will not participate in the reaction). Common polar protic solvents are DMF, DMSO, THF and acetonitrile.

[0160] According to the research results and theories of previous researchers, it can be speculated that the main products of the photo-cleavage reaction of Boc-Phe-CPPG compounds are CPPG protective groups and free Boc-Phe-OH. Therefore, for the sake of comparison, Boc-Phe-CPPG, CPPG and Boc-Phe-OH were subjected to HPLC respectively before photo-cleavage, and the retention time of each component in HPLC was compared to analyze which product was produced after photo-cleavage.

[0161]

[0162] Then the concentration is set to 10 -2 Take 3 mL of mol / L Boc-Phe-CPPG solution (solvent is THF:H2O=4:1) and put it into a 10 mL quartz test tube (glass absorbs ultraviolet light, which is not conducive to the reaction). Place it in a photochemical reactor, stir at room temperature, and irradiate with a high-pressure ultraviolet mercury lamp (1000W). Take a sample every hour for HPLC. After 9 hours of reaction, the raw material peak disappears.

[0163] Subsequently, the HPLC retention time of the previous standard was compared ( Figure 4), it can be preliminarily determined that the photocleavage product is indeed CPPG protecting group and free Boc-Phe-OH, and the TLC spot plate results and high-resolution mass spectrometry data of the reaction solution also confirmed this result ( Figure 5 ). Boc-Phe-OH:HRMS(ESI)m / z calcd for C 14 H 19 NO4Na + (M+Na) + 288.12063, found 288.12057. CPPG:HRMS(ESI)m / z calcd for C 22 H 17 O5P + (M+Na) + 415.07058,found415.07034.

[0164] Since the reaction rate in THF solvent is not satisfactory, the present invention conducted a comparative experiment with four mixed solvents (DMF / DMSO / THF / acetonitrile: H2O=4:1) to prepare Boc-Phe-CPPG solutions with a starting concentration of 5×10 - 3 mol / L (too low a concentration will result in too fast a reaction and difficult HPLC monitoring, while too high a concentration will result in too long a reaction). Take 3 mL of each and put them into a 10 mL quartz test tube. Stir them in a photochemical reactor at room temperature and irradiate with a high-pressure ultraviolet mercury lamp (800 W). Finally, it was found that the photoshearing effect of the acetonitrile mixed solution was better. Therefore, under the above conditions (initial concentration 5×10 -3 mol / L, 800W, room temperature) with acetonitrile as mixed solvent, the photoshearing reaction kinetics was studied. Figure 7 As shown, from top to bottom are HPLC graphs of 0, 0.5, 1.5, 2.5, 3.5, and 4.5 hours of illumination (50 μL is accurately sampled each time). As the reaction proceeds, the peak height of the raw material Boc-Phe-CPPG gradually decreases. The peak of the product is not obvious, probably due to the low concentration and poor signal response of the product.

[0165] According to the peak height of the raw material CPPG-Phe-Boc in the HPLC spectrum of each time period, the residual concentration of the raw material in each time period can be roughly calculated (the peak height of each time period divided by the initial peak height, multiplied by the initial concentration), as shown in Table 1, and the kinetic curve of the photoshearing reaction can be drawn based on this data. Figure 7 shown.

[0166] Table 1 Peak Height and Concentration of Raw Material in Each Period

[0167]

[0168] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should be included in the protection scope of the present invention.

Claims

1. A novel photocatalytically cleavable carboxylic acid protecting group 2-nitro-phosphoryloxybenzyl compound and its amino acid derivatives, characterized in that: The molecular structure formula of 2-nitro-phosphoryloxybenzyl compound 1 and its amino acid derivative 2 is:

2. The method for preparing the novel photocatalytically cleavable carboxylic acid protecting group 2-nitro-phosphoryloxybenzyl compound and its amino acid derivative according to claim 1, characterized in that: The following steps are involved: Step 1: using vanillic aldehyde or vanillin ketone as a raw material to react with (R)2POCl or POCl3 respectively, adding a base to a solvent A to catalyze the reaction, after the reaction is completed, recovering the solvent by rotary evaporation, extracting the residual solution with ethyl acetate and separating the aqueous phase, combining the organic phases and washing them with deionized water for multiple times; separating and purifying to obtain a phosphoryloxy vanillic aldehyde compound or a phosphoryloxy vanillin ketone compound; Nitrate the obtained phosphoryloxy vanillic aldehyde compound or phosphoryloxy vanillic ketone compound with nitric acid, separate and purify to obtain 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic aldehyde ketone compound; Step 2: using the 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic aldehyde ketone compound obtained in step 1 as a raw material, and using sodium borohydride as a reducing agent to carry out a reduction reaction in a solvent B. After the reaction is completed, concentrated hydrochloric acid is added to adjust the pH value to 2-3 to terminate the reaction, and the solvent is recovered by rotary evaporation. The residual solution is extracted with ethyl acetate and separated from the aqueous phase, and the organic phases are combined and washed with deionized water for multiple times; then separation and purification are performed to obtain the corresponding derivative product 2-nitro-phosphoryloxy vanillic aldehyde compound or 2-nitro-phosphoryloxy vanillic ketone compound, 2-nitro-phosphoryloxy benzyl alcohol compound; Using 2-nitro-phosphoryloxybenzyl alcohol compounds as raw materials, reacting with concentrated hydrohalic acid under the catalysis of anhydrous zinc halide, after the reaction is completed, recovering the solvent by rotary evaporation, extracting the residual solution with ethyl acetate and separating it from the aqueous phase, combining the organic phases and washing them with deionized water for multiple times; and then separating and purifying to obtain 2-nitro-phosphoryloxybenzyl halogen compounds; Releasing a 2-nitro-phosphoryloxybenzyl halogen compound with sodium amide or sodium hydrosulfide to convert it into a corresponding 2-nitro-phosphoryloxybenzylamine compound or a 2-nitro-phosphoryloxybenzylthiol compound; Step 3: The 2-nitro-phosphoryloxybenzyl alcohol compounds, 2-nitro-phosphoryloxybenzyl halogen compounds, 2-nitro-phosphoryloxybenzyl amine compounds or 2-nitro-phosphoryloxybenzyl thiol compounds obtained in step 2 are respectively subjected to coupling reaction with Boc- or Fmoc-protected amino acids (PG-AA-OH), and their corresponding amino acid derivatives 2 are obtained after separation and purification.

3. The method for preparing the novel photocatalytically cleavable carboxylic acid protecting group 2-nitro-phosphoryloxybenzyl compound and its amino acid derivative according to claim 2, characterized in that: The feed ratio of (R)2POCl or POCl3 to vanillic aldehyde or vanillic ketone is 1 to 4:1, the reaction temperature is -5 to 50°C, and the reaction time is 1 to 5 hours; The feeding ratio of the nitric acid to the phosphoryloxy vanillic aldehyde compound or the phosphoryloxy vanillic ketone compound is 1:1-3, the reaction temperature is -5-50°C, and the reaction time is 1-5 hours.

4. The method for preparing the novel photocatalytically cleavable carboxylic acid protecting group 2-nitro-phosphoryloxybenzyl compound and its amino acid derivative according to claim 3, characterized in that: The separation and purification comprises: extracting with ethyl acetate and separating with water phase, combining organic phases and washing with deionized water for 2-3 times; dripping an alkane or ether solvent with low polarity into the ethyl acetate solution until crystals or precipitations are formed, and completing the separation and purification by filtering and washing or recrystallization; The solvent A is one or more of tetrahydrofuran, acetonitrile, benzene, toluene, chloroform, dichloromethane, N,N-dimethylformamide, and N-methylpyrrolidone; The base is one or more of tertiary amines TEA, DIEA, NMM, pyridine and DMAP.

5. The method for preparing the novel photocatalytically cleavable carboxylic acid protecting group 2-nitro-phosphoryloxybenzyl compound and its amino acid derivative according to claim 4, characterized in that: The reaction conditions of the base-adding catalytic reaction and the sodium borohydride reduction reaction are: stirring at room temperature for 2 to 3 hours; The number of times of washing with deionized water is 2 to 3 times; The B solvent is one or more of alcohols, acetonitrile, or tetrahydrofuran; The alkane or ether solvent is one or more of petroleum ether, n-hexane, cyclohexane, diethyl ether, or n-butyl ether.

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