Abstract: This paper introduces the preparation and application progress of protective amino acids ——— raw materials for peptide solid-phase synthesis reaction, discusses in detail the α-amino group, α-hydroxyl group and various new protective groups of side-chain active groups of amino acids, summarizes the structural characteristics and application conditions of new protective amino acids, and looks forward to the future development direction of protective amino acids.
Key words: Solid-phase peptide synthesis amino acid protection
In 1962, Merrifield founded the solid-phase peptide synthesis method [1], and since then, solid-phase synthesis using protective amino acids as raw materials has attracted attention for its unique advantages. In the following 10 years, the solid-phase synthesis of peptides has been continuously improved, from small-scale and short peptide chain synthesis to large-scale and long peptide chain synthesis. The anti-AIDS drug T-20, launched in 2003, is a small peptide drug composed of 36 amino acid residues that are fully synthesized. In the 90s of the last century, after the combination of combinatorial chemistry and peptide synthesis technology, the synthesis of combinatorial peptide libraries appeared, which played a huge role in promoting chemistry, biochemistry, medicine, molecular biology and other fields. In the process of development, the requirements for the protection of amino acids have also been continuously improved.
In the synthesis of solid-phase peptides, the active functional groups of the raw material amino acids participating in the reaction are protected, and the protective groups of these active groups can be removed after the reaction is completed.
When selecting amino acid protection groups, the problem of reaction selectivity should be fully considered, that is, α-amino protection groups and α-carboxyl protection groups are temporary protective groups, and an amino acid should be immediately removed after binding to the peptide chain. The side-chain protection group is a "permanent" protective group that is not affected by reactants during the synthesis of peptides, does not react, and is finally removed after peptide synthesis is completed. Therefore, the stability of the side chain protection group is greater than that of the α-amino protection group and the α-carboxyl protection group.
The selection of protective amino acids is directly related to the yield and purity of synthetic products, and the by-products and racemic formed in the reaction bring great difficulties to purification, so the development of new protective amino acids is an important part of solid-phase peptide synthesis.
At present, amino acid protection is mainly divided into three categories: α-amino protection, α-carboxyl group protection and side-chain active group protection. Based on these three classifications, this paper focuses on some new varieties of amino acid protection in recent years, as well as their structural characteristics and application conditions.

1 α-amino protection

In principle, amino groups can be reversible shielding by acylation, alkylation, alkylation and other reactions. Protective groups based on sulfur and phosphorus derivatives have also been reported. In the past 10 years, hundreds of different amino protective groups have been developed, mainly including alkoxycarbonyl protective groups, acyl protective groups and alkyl protective groups. The reported results suggest that there is no universal ideal amino protection group for peptide solid-phase synthesis.
1.1 Alkoxycarbonyl (carbamate) protective groups
This type of protective group is combined with amino acids in the form of:

Thereinto

a: Benzyloxycarbonyl(Z); b: tert-butoxycarbonyl (Boc); c:9-fluoroxycarbonyl (Fmoc); d:2-(4-nitrobenzenesulfonyl)ethoxycarbonyl (NSC); e:1,1-dioxyphenyl (o)2-thiophenemethoxycarbonyl (Bsmoc); f:2-(methanesulfonyl)-3-phenyl-2-propyloxycarbonyl (MSPOC); g:2-(tert-butylsulfonyl)-2-propyloxycarbonyl (Bspoc) :h:cyclopentanoxycarbonyl (Poc)
Z, Boc, and Fmoc are the three most common amino protection groups. Z is a long-lasting amino protective group that is still widely used today. Its advantages are easy to prepare: the resulting Z-amino acids are easily crystallized and stable; It is not easy to racema during activation; The protective group can be removed under H2/Pd, HBr/AcOH, Na/liquid ammonia, etc. [2]. BOCs can be used in most peptide synthesis coupling processes and can be removed under acid hydrolysis conditions while avoiding the effects of catalytic hydrogenation, alkaline hydrolysis, and sodium/liquid ammonia reduction. Boc is usually removed under anhydrous TFA, 0°C reaction conditions [2]. FMOC is the only carbamate-type amino acid protection group that can be dissociated under weak alkali conditions, and FMOC deprotection can be done at room temperature using urine-piperidine solution or diethylamine DMF solution [3].
With the deepening of the understanding of amino protection, some amino protection groups with better selectivity or stability were obtained by modifying the functional groups on the basis of the above three protective groups.
NSCs are considered a better protective group than FMOC. NSC is more stable under alkaline conditions and less sensitive to weakly alkaline solvents. NSC is more hydrophilic than FMOC, and the possibility of asparagine formation is further reduced [4]. Consistent with FMOC, NSCs form paraffin in a deprotection reaction and eventually react with piperidine addition. However, in the Fmoc reaction, the formation of diphenylfulpenide adducts is reversible, while the adducts formed by the NSC reaction are irreversible, which reduces the probability of side reactions [5]. NSC can be removed at room temperature with piperidine, 2-aminoethanol, morpholine, liquid ammonia, etc.
Carpino et al. developed a series of novel alkali unstable amino protection groups Bsmoc, Mspoc, and Bspoct [6~8]. This type of group dissociation requires the addition of a nucleophile (such as a secondary amine) accompanied by the release of carbamates. BSMOC is stable to acids and tertiary amines, but is prone to dissociation when secondary amines are present. Since the deprotection process is also an elimination reaction, side reactions rarely occur.
Poc is a different amino protection group from Boc and Fmoc, which is stable for both acids and bases, Surajit et al. found that the Poc group is still stable after reacting with trifluoroacetic acid at 25°C for 1 h, while the side chain protection group such as tert-butyl can be completely removed, and the side chain protection group can be selectively removed by using this feature [9]. The Poc group can be removed with tetrasulfomolybdate under ultrasonic agitation, and can also be removed by HBr/AcOH or Na/liquid ammonia.
1.2 Acyl type protective groups
There are two main types of protective groups: carboxylyl and benzenesulfonyl, and the forms after combining with amino acids are as follows:

a: trifluoroacetyl group (TFA); b: p-toluene sulfonyl (Tos); c: Proximal nitrobenzene sulfonyl group (oNbs)
TFA was first introduced by Weygand in 1952 for peptide synthesis [2], and its removal conditions are relatively mild, and it can be easily removed by piperidine or sodium hydroxide. However, it is easy to racema during activation and easy to break the chain during alkaline hydrolysis, so it is widely used.
Tos is also an early protective group [3], which has very good stability and can only be removed by Na/liquid ammonia treatment, and is not affected by acid-base or catalytic hydrolysis. However, due to the troublesome operation of Na/liquid ammonia treatment and the breakage of some peptide bonds, it is rarely used at present.
The situation was greatly improved after the introduction of orthonitro to the benzene ring of Tos and the obtaining oNbs [10]. The sulfonamide protection group allows the N-acylation of amino acids without the racemic caused by the pyroxone mechanism. And this protective group can be removed by treatment with phenylthiophen or alkane mercaptan, which overcomes the shortcomings of Tfa and Tos that are not easy to remove.
1.3 Alkyl protective groups

a: triphenylmethyl (Trt); b: N-1- (4, 4-dimethyl-2, 6-dioxanexyl)ethyl (Dde); c: N-1- (4-nitro-1,3-dioxinde-2-ene) ethyl (Nde)
TRT is a commonly used alkyl-type protection group [2], which can be removed with anhydrous HCI/MeOH, TFA, or HBr/AcOH under mild conditions. Because Trt has a large steric steric hindrance, it is generally difficult to introduce and bind to amino groups, and the Trt on the side chain is more stable than the Trt on the main chain, so the application of Trt is mostly used for the protection of amino acids in the side chain, while the protection of α-amino groups is less applied.
Dde and Nde are novel alkyl amino protection groups. Dde is very stable to TFA and piperidine, and it needs to be removed with 2% (v/v) hydrazine/DMF, so if the appropriate deprotection conditions are selected, the amino protection on the main chain can be retained, and the amino protection group on the side chain can be removed, and then branched-chain peptides can be synthesized.
Barrie Kellam et al. studied the characteristics of Dde, and on this basis, the resulting Nde showed good acid and secondary/tertiary base stability [11]. Similar to Dde, the structure of NDE cannot form azoledone, which effectively reduces racemic and can be removed by 2% (v/v) of hydrazine/DMF deprotection. The addition of indenium group can not only enhance the nucleophilic attack, but also improve the UV absorption value, which is conducive to online detection.

2 α-carboxyl protection

Solid-phase synthesis of peptides - generally does not require the protection of α-carboxyl groups, but only under rare conditions, it is necessary to protect α-carboxyl groups. For example, synthetic glutathione requires the γ-carboxyl group of glutamate to react, while the α-carboxyl group needs to be protected. At present, hydroxyl protection groups are roughly divided into two categories: ester groups and acylhydrazines.
2.1 Ester group
Ester protection of hydroxyl groups is the most commonly used method, mainly of the following types:

a: Me ester ; b: ethyl ester (Et); c: tert-butyl ester (tBu); d: benzyl ester (Bzl); E:2, 4-Dimethyloxyphenylmethyl ester (2, 4-DMB)
Among them, Me, Et and Bzl are prepared in simple ways and can be removed by alkali saponification. tBu is one of the most commonly used carboxyl protection groups. tBu is more stable than Me and Et, and is not easily saponified by alkali, but can be detoxified by HCI, HBr/AcOH, TFA, HF and other acids. Acid-sensitive ester 2, 4-Dmb can be removed by treatment with 1% TFA/CH2Cl2 without causing other side chain protective shedding, and this selectivity can be used in some special cases, such as the preparation of cyclic peptides by combining side chain hydroxyl groups with backbone amino groups [12].
2.2 Hydrazine
Acyl hydrazine is also used for the protection of Cα or side-chain carboxyl groups, but in this case, further azide coupling is necessary, less commonly used in solid-phase peptide synthesis. Its form is generally NH2CHRCONHNHR'.

3. Side chain active group protection

3.1 Ω-carboxyl protection group
Aspartic acid (Asp) and glutamic acid (G1U) have two carboxyl groups, and the omega-carboxyl groups on the side chain are prone to side reactions under the action of alkali or strong acid. For example, the lateral chain ester formed by aspartic acid during activation is generated by the corresponding intermediate state of succinimide to form isoparagine peptide. Commonly used protective groups are the tert-butyl ester group (OtBu) used in the Fmoc strategy and the β-cyclohexyl ester group (OcHex) used in the Boc strategy. These two protective groups can usually inhibit the occurrence of these side reactions, but when Asp is connected to Gly, Thr (tBu), and Cys (Acm), the side reactions are very violent and must be protected by N-2-hydroxy-4-methoxyphenyl (Hmb) [13]. When removing the protective group, TFA can be used to capture carbocations with mercaptans to reduce the occurrence of side reactions.
Karlstr⌀m and Undén synthesized two protective groups, β-2,4-dimethyl-3-amylester (ODmp) and β-3-methylpenta-3-ester group (OMpe) [14], [15]. It was found that ODmp and OMpe could effectively inhibit the asparagine conversion of aspartic acid, and could prevent the formation of asparagine more than OtBu and OcHex, which could be used for the synthesis of difficult fragments.

3.2 Hydroxyl protection
The phenolic hydroxyl group of tyrosine (Tyr), serine (Ser), and hydroxyl group of threonine (Thr) are all reactive groups that must be protected during the solid-phase synthesis of peptides. Commonly used groups are 2-bromophenoxycarbonyl (BrZ), benzyl (Bzl), and tert-butyl (tBu) used in the Fmoc strategy. BrZ mainly protects the phenolic hydroxyl group of Tyr, which is very stable to nucleophiles and TFA, and needs to be removed with strong acids. It is extremely unstable to bases and completely dissociated in 20% piperidine. Bzl can be removed by hydrolysis, HF, Na/liquid ammonia, and there will be partial dissociation in TFA. tBu can be removed by TFA (reaction at room temperature for 2 h), HCI/TFA or concentrated HCl at 10°C for 10 min.
Rosenthal et al. used 2,4-dimethylpentaoxycarbonyl (Doc) as the protective group of Tyr, which had a good protective effect under the Boc strategy. Doc is 1 000 times more stable than BrZ, and can be completely eliminated by HF [16].
Compared with Bzl, cHex is more stable in 50% TFA/DCM and does not form rearrangement by-products, and is also stable to 20% piperidine, so it can be used not only in Boc strategy but also in Fmoc strategy, but it is more difficult to synthesize [17].
Based on the study of all the above groups, József Bódi et al. investigated a ring-opening analogue of cHex: 3-pentanyl (Pen). It is stable not only in 50% TFA/DCM but also at 20% piperidine/DMF, so it can be used in both Boc and Fmoc strategies, and is easier to synthesize than cHex [18]. The removal of the Pen group was carried out by the HF method.

3.3 Indole protection
Tryptophan (Trp) generally does not require side-chain protection in FMOC strategies, but the tert-butylation and sulfonation of indole will intensify when other side-chain amino acid protection groups are finally removed. Boc group protection is a good way to prevent these side effects.
When the Boc strategy synthesizes Trp-containing peptides, indole is prone to cyclization or dimerization, so the indole part must be protected. Usually protected with a Nin-formic acid group (For), which is stable for treatment of repeated TFA and can be removed by alkali or HF/mercaptan. However, it also brings other problems, such as odor, polymerization side reactions of mercaptans. In addition, the Z group and 1, 3, 5-trimethylbenzene sulfonyl groups (MTS) can be used to protect Trp. However, when the Z group is removed by HF deprotection method, the reaction is incomplete. The Mts group will be gradually decomposed by TFA during peptide synthesis.
Yuji Nishiuchi et al. summarized the characteristics of Z and Mts and introduced the cyclohexyloxycarbonyl (Hoc) group. The dissociation of the carbamate-type Hoc group will form a carboxylate intermediate state, which will reduce the nucleophilic attack on the Trp indole ring [19]. In addition, the carbon ring on the Hoc group can increase the stability of bases and acids. The experimental results show that Hoc is very stable when the peptide chain is elongated and can be removed by HF without the use of thiols.

3.4 Sulfhydryl protection
The sulfhydryl groups in cysteine have the characteristics of strong nucleophilicity, easy oxidation and acidity, which require the selective shielding of sulfhydryl groups in all synthesis processes. The commonly used protective groups are Trt, tBu, and acetamide methyl group (Acm), and the deprotection conditions are different. Trt is the most widely used protective group, which can be removed by HF, TFA, I2, Hg (II.), etc.; tBu can be removed by TFMSA: Acm can be removed by I2 and Hg (II.). Modifications on the basis of the above groups yield similar tert-butyl sulfuryl (StBu) [20] and trimethylacetamide methyl (Tacm) [21], which increase the stability of these two groups and make the preparation method simpler. By taking advantage of the differences in the deprotection conditions of each protective group, the formation of disulfide bonds can be selective.
Sulfonylnitropyrimidine (NPYS) is a novel sulfhydryl protection group [22] that is unstable to the base and cannot be used in the FMOC strategy. However, if the Boc strategy is adopted, the cysteine it protects can be introduced into the N-terminus, and under weak alkaline conditions, it can be removed, so that the cyclization reaction can be carried out conveniently.

3.5 Imidazole protection
Histidine (His) is one of the most problematic amino acids in peptide synthesis. When the imidazole ring of histidine is not protected, there are two main problems: N-acylation and racemic. Reversible shielding of the azole functional group avoids these problems. The early protection groups used in the Boc strategy were Boc and Tos. The Boc group will be removed at the same time as the α-amino protection group, resulting in acylation and racemic in each step of the condensation reaction, so it is rarely used at present. Tos is the most common His protection group in the Boc strategy. It has good solubility and is easy to remove, and can be treated with HF or TFMSA. However, the Tos group is also deficient, and it will be partially removed under the action of HOBt, so when HOBt/DCC is used, side reactions such as benzosulfonylation will occur.
The benzyloxymethyl (Bom) group compensates for the above group deficiency [23], and Bom can completely shield the π-nitrogen atoms caused by racemicization by the imidazole group, and is also stable under condensation reaction conditions, and can be removed by HF or TFMSA treatment.
In the FMOC strategy, the most commonly used protective group of histidine azole is Trt. TRT shields the π-nitrogen atom of the imidazole group, which can effectively inhibit racemic. Although the reaction is relatively slow due to the large steric resistance of Trt, the steric hindrance of Trt can effectively reduce the rearrangement of alkali catalysis and reduce the occurrence of side reactions. TRT is very stable in the process of peptide synthesis, and can be easily removed with 95% TFA after synthesis.
Nπ-allyl (All) and Nπ-allyl oxymethyl (AIom) [24] are a novel class of protective groups. All and Alom act on the π-nitrogen atoms of the imidazole group, which can inhibit racemic. At the same time, it is stable to acids and bases, and can be used in Fmoc and Boc strategies, and is relatively easy to prepare, and can be removed with nucleophiles and palladium.

3.6 ε-Amino Protection
Lysine (Lys) is highly ε-aminobasic and nucleophilic at the same time. In the Boc strategy, it is usually protected by 2-chlorobenzooxycarbonyl [Z(2-Cl)], which has good acid stability and can prevent the phenomenon of early removal of the side-chain protection group during the process of dissociation of the Boc group. In the FMOC strategy, the Boc group is a good protective group, which has good stability to alkali and can effectively inhibit the occurrence of side reactions.
The protective groups such as Npys, Dde, and Tfa introduced earlier can also be used to protect Lys, Npys, and Tfa against piperidine instability, which are generally used in Boc strategies, and Dde must be eliminated by hydrazine detoxification, which can be used for both Fmoc and Boc strategies.
The Dde group also has its limitations, and when synthesizing long peptides, there will be partial dissociation and migration phenomena under the action of piperidine. The monomethoxytriphenylmethyl (MMT) and dimethoxytriphenylmethyl (DMT) synthesized by Sefan Matysiak et al. have enhanced stability to bases and can be used in FMOC strategies [25]. At the same time, Mint and Dmt are extremely unstable to acids, and can be removed by acetic acid/trifluoroethanol/dichloroethane (1∶2∶7, v/v) without affecting other side chain groups, so they can be used for the synthesis of cyclic peptides and branched chains.
3.7 Guanidine protection
Although the guanidine arginine group is highly basic and protected by protonation under normal reaction conditions, the corresponding activated ester has low solubility in organic solvents during activation, which is not conducive to peptide synthesis. Under some conditions, guanidine groups are also partially acylated, so protection is necessary.

Groups such as 4-methoxybenzenesulfonyl (MBS), Mts, Tos, 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl (Mtr), 2, 2, 5, 7, 8-pentamethylseman-6-sulfonyl (PMC), and 2, 2, 4, 6, 7-pentamethyl dihydrobenzofuran-5-sulfonyl (Pbf) are commonly used arginine guanidine protection groups in solid-phase synthesis [3]. The acid instability increased, in the order of Tos<Mbs< Mtr<Pmc< Pbf, and Pbf showed the best deprotection kinetics. As a temporary protective group, Mts have good compatibility with Boc, and unlike Tos, Mts can be removed by trifluoromethane sulfonic acid (TFMSA)/TFA/benzosulfide. MTR is commonly used in Fmoc strategies, but requires a longer time to deprotect with TFA/anisulfide. The acid instability of the PMC group is similar to that of the Boc group. Therefore, Boc (e.g., protective Lys) and Pmc or Pbf (protective Arg) can be optimally combined as side-chain protection groups.
3.8 Miscellaneous
3.8.1 Enzymatic hydrolysis of protective groups
When synthesizing acid-unstable or base-unstable peptide derivatives and peptide conjugates, the protective groups of amino acids need to be removed under mild or even neutral reaction conditions. Glycopeptides, phosphopeptides, and lipopeptides, as well as sulfate-containing peptides, are among these sensitive compounds. Treatment of these derivatives with alkali often results in β-elimination reactions or other adverse side reactions of Ser and Thr derivatives, while treatment with acids leads to degradation of the non-peptide fraction of the conjugate in some cases. The application of enzymatic hydrolysis of protective groups can solve the above problems.

Tetraphenylglucooxycarbonyl (BGloc) is a carbamate-type enzymatic hydrolytic protection group [26], which contains O-benzyl-protected glucose as a carbohydrate moiety, which attaches carbamate oxygen atoms. After the benzoyl group is removed, the mixture of α-glucosidase from baker's yeast and β-glucosidase from almonds breaks the glucosidic bond, thereby releasing carbamate. It can be used to protect amino groups.
Phenylhydrazine (PH), studied by Gernot H.M. et al., is also an enzymatic hydrolytic protection group [27], which can be used to protect carboxyl groups, and can be used to generate acyldiazene under the action of enzymes and oxygen using tyrosinase extracted from mushrooms, and then completely removed by hydrolysis.
3.8.2 Photolytic protection groups
In 1991, Fodor introduced the photolytic protection group o-nitro-3, 4-dimethoxybenzocarbonyl (NVOC) as an amino acid protection group for the first time, but the photolysis rate of NVOC is very slow, 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) is a new type of photolytic protection group, Kumar R. Bhushah et al. used it for the protection of amino groups, and found that under ultraviolet irradiation, It dissociates 2 times faster than NVOC [28].

4. Discussion and outlook

Protective amino acids are key raw materials for peptide synthesis, and the protection strategies vary according to the different methods of synthesizing peptides.
The development of protective amino acids mainly focuses on: (1) the development of new α-amino protection groups, such as Nsc, Dde, and Nde, which have the characteristics of reducing racemic and improving the deprotection efficiency; (2) Development of protection of active groups in the side chain, a variety of protective groups with excellent stability, high selectivity and reduced occurrence of side reactions were developed for different active groups. Protective amino acids prepared in real-world applications have shown excellent performance.
With the increasing complexity of the structure of synthetic peptides, the protective groups of amino acids tend to develop in the direction of selectivity and specificity, and the enzymatic and photolytic protective groups may become new hot spots in future research due to their unique advantages.

The synthesis of tirzepatide mainly adopts the strategy of combining solid-phase synthesis (SPPS) and liquid-phase synthesis (LPPS), taking into account both efficiency and purity.

Basic Info
Name：Tirzepatide
Sequence：H-Tyr-{Aib}-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-{Aib}-Leu-Asp-Lys-Ile-Ala-Gln-{diacid-C20-gamma-Glu-(AEEA)2-Lys}-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2
Molecular Formula：C225H348N48O68
Mol. wt.：4813.45

Synthesis method

The synthesis of tirpotide mainly adopts the strategy of combining solid-phase synthesis (SPPS) and liquid-phase synthesis (LPPS), taking into account efficiency and purity.
Solid-phase/liquid-phase combined synthesis method
Eli Lilly: The dual agonist of glucose-dependent insulinotropic peptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor developed by Eli Lilly is to synthesize multiple short peptide fragments (AA1-14, AA15-21, AA22-29 and AA30-39 fragments) by solid-phase synthesis, as shown in Figure 1 below. The final product is obtained by combining the above four fragments by liquid phase synthesis method. In the process of solid-phase synthesis of these short peptide fragments, multiple long fragments are obtained by coupling dipeptides or tetrapeptides with Fmoc or Boc protection. This reduces the amount of amino acids used while reducing the formation of impurities, improving production efficiency and product quality.

The segmented synthesis strategy of fully protected peptides was used to synthesize fully protected peptides (e.g., 1-17 and 18-39) in solid phase, and then deprotected and conjugated through liquid phase deprotection, which significantly shortened the synthesis cycle and improved the yield.
Solid-phase synthesis method
Amino acids are stepwise coupled using Rinkamide resin as a solid-phase carrier. For example, Fmoc-Ser(tBu)-OH is first fixed, then unprotected (piperidine/DMF) and then conjugated (HOBt/DIC activated carboxyl groups) until the target sequence is completed. Solid-phase synthesis method: After the synthesis, the resin is lysed with trifluoroacetic acid (TFA) and the side-chain protection group is removed to obtain crude peptides. Fragment condensation and natural chemical linkage: For long peptide chains, pre-synthesized short peptide fragments can be used for liquid phase coupling, such as by thioester bonds (e.g., NCL technology).

Key Synthesis Steps

1. Amino acid activation and conjugation Use activation reagents such as HOBt, HATU, and DIC to activate carboxyl groups and condense with free amino groups to form peptide bonds. For example, complex amino acids such as Fmoc-Lys (aeea-aeea-γGlu(α-OtBu)-eicosanedioic tBu ester)-OH need to be activated step by step. Control the reaction conditions (temperature, pH, time) to reduce racemic and side reactions7.
2. Protective base strategy Fmoc/tBu strategy: the amino group is protected by Fmoc, and the side chain (such as the hydroxyl group of Ser and Thr) is protected by tert-butyl (tBu)7. BOC/BZL Strategy: Applies to side-chain protection for specific amino acids.
3. Lysis and crude peptide preparation The lysate is usually a DCM solution containing TFA (20% TFE), which is preliminarily purified by ether precipitation after lysis.

Purification process

Optimizing impurity control in tirpotide synthesis is a challenge and requires a combination of advanced purification technologies:
Functionalized silica gel separation column: 3-chloropropyltriethoxysilane and dimeraminocyanocyano/acetazolamide modified silica gel to enhance adsorption selectivity for target peptides and improve separation efficiency.
Combined with modified diatomaceous earth (levulinic acid treatment) to further remove hydrophobic impurities.
Chromatographic purification: Reversed-phase high-performance liquid chromatography (RP-HPLC, C18 and C8 columns) and monodisperse polymer reverse packing Seplife RP LXMS 15 (300) are used for fine purification of the end product, ensuring a purity > 98%.

Optimization direction of synthesis process

Fragment design and connection efficiency: Optimize the fragment length (e.g., 1-17 and 18-39) and coupling conditions for solid-phase synthesis to reduce side reactions.
Biosynthesis technology: Using genetically engineered strains to express tirpotide, combined with chemical synthesis and modification, reduce costs.
Separation Technology Upgrade: Introduce nanofiltration, ultrafiltration and other technologies to simplify the post-processing process and increase production capacity.

Challenges and prospects

Impurity control: Impurities (such as missing peptides and oxidation products) in the solid-liquid mixing method are difficult to completely remove, and the purification process needs to be continuously improved. Market competition: Eli Lilly's tirpotide is 20% lower than semaglutide due to its flexible chemical synthesis path and sufficient production capacity, which puts pressure on domestic GLP-1 drug companies. Indication expansion: Tirpotide has been approved for the treatment of obstructive sleep apnea, and may be expanded to heart failure and other fields in the future, promoting further optimization of the synthesis process.

Summary

The synthesis process of tirpotide is based on the solid-liquid combination method, combined with high-efficiency activation reagents and functionalized purification technology, balancing cost and purity. In the future, through the integration of biosynthesis and chemical synthesis and the development of new separation materials, its process efficiency and product quality are expected to be further improved, supporting its continued expansion in the global diabetes and obesity treatment market.

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Linear cell-penetrating peptides can improve and enhance the delivery of oligonucleotide drugs, but linear peptides are still unstable to proteolytic enzymes and may limit oligonucleotide delivery and efficacy and safety. Cyclization of cellular transmembrane peptides can improve their stability and improve the efficiency of oligonucleotide drug delivery.
The method of preparing cyclic peptide-oligonucleotide conjugates is mainly by liquid phase connection. That is, linear peptides are synthesized first, then the linear peptides are cyclized to form cyclic peptide fragments, and then the cyclic peptide fragments and oligonucleotide fragments are connected to form cyclic peptide-oligonucleotide conjugates through chemoselective reactions. In addition, cyclized cell-osmotic peptides often contain multiple positively charged arginine or lysine, which will aggregate or precipitate with negatively charged oligonucleotides during reaction. The solid-phase method can avoid this problem of aggregation and precipitation.
As with the preparation of linear peptide-oligonucleotide conjugates by solid-phase synthesis, the destruction of oligonucleotides by trifluoroacetic acid also needs to be considered when preparing cyclic peptide-oligonucleotide conjugates by solid-phase synthesis, so it is also necessary to avoid the use of trifluoroacetic acid. Another important issue considered in the preparation of cyclic peptide-oligonucleotide conjugates by solid-phase synthesis method is the cyclization of peptide fragments. The simplest way to cyclize polypeptides is to ring with dibrominated small molecules by sulfhydryl groups, which is a fairly mature reaction at the peptide level, but cyclic peptide-oligonucleotide conjugates include not only polypeptides, but also oligonucleotides, and it is best to cyclize with the presence of sulfhydryl groups, so it is necessary to prepare cyclic peptide-oligonucleotide conjugates containing sulfhydryl groups. The sulfhydryl protection group of hemiphotoine is generally triphenylmethyl (Trt), but triphenylmethyl (Trt) finally needs to be removed from trifluoroacetic acid, which is definitely incompatible with solid-phase synthesis systems. Therefore, a suitable cysteine sulfhydryl protection group needs to be found to prepare cyclic peptide-oligonucleotide conjugates. StBu can form a disulfide bond with cysteine to protect cysteine, and only a reducing agent can remove this protective group. There is no reducing agent involved in the synthesis of solid-phase peptides and solid-phase oligonucleotides, so in theory, StBu exists stably in these two solid-phase synthesis processes, and Fmoc-Cys(StBu)OH can be used to synthesize amino acids.
[Solid-phase condensation of cyclic peptide-oligonucleotide conjugates, liquid-phase ringing]
The operation steps of cyclic peptide-oligonucleotide conjugates before ring formation are the same as those of linear peptide-oligonucleotide conjugates, which are first carried out for peptide synthesis and then for the synthesis of solid-phase oligonucleotides. First, the resin needs to be connected to a small C7-NH2 molecule, the N-terminus of this small molecule is protected by Fmoc, where peptide synthesis can be carried out, hydroxyl group is protected by DMTr, and solid-phase oligonucleotide synthesis can be carried out here. Then, the Fmoc protective group on the small molecule was removed with 20% piperidine, and the remaining amino acids were sequentially coupled to the C7-NH2 molecule. The amino groups of tryptophan, histidine, arginine and lysine side chains need to be protected with Boc, the hydroxyl groups of serine, tyrosine, threonine side chains are protected with TBS, and the sulfhydryl groups of cysteine are protected by StBu. The end of peptide synthesis requires the drying of the solid-phase carrier and then the synthesis of solid-phase oligonucleotides. After the synthesis, the polypeptide-oligonucleotide conjugate containing cysteine is cut off from the resin directly with ammonia. At this time, according to the design of the experiment, the protective group should continue to retain on the peptide-oligonucleotide conjugate, without purification, the StBu protective group of cysteine acid can be completely removed by dissolving the reaction with TCEP solution for 1 hour, purifying and lyophilizing and 1,3-di(bromomethyl)benzene in the solution for ring formation, and monitoring the reaction process by high-performance liquid chromatography (HPLC), generally one hour can be successfully cyclized.

【Monacyclic peptide-oligonucleotide conjugates】
There is only one cysteine, which cannot be cyclized and generally needs to contain 2 cysteines. The synthesis steps are the same as those described above. After removing the two StBu protective groups on the peptide-oligonucleotide conjugate, cyclic peptide-oligonucleotide conjugates can be synthesized by using the principle of 1,3-dis(bromomethyl)benzene and two sulfhydryl groups in solution.

In addition to di(bromomethyl) benzene, ring-forming linkers can also be other types of linkers. Monacyclic peptide-oligonucleotide conjugates can also be synthesized from alkyl olefins and biphenyls, the difference is mainly that in the last step of ring formation, alkyl olefins and biphenyls are used to replace di(bromomethyl)benzene, and the reaction time is also 1 hour.
[Synthesis of bicyclic peptide-oligonucleotide conjugates]
In addition to monocyclic peptides, there are also bicyclic peptides, which react with two cysteines and 1,3-dis(bromomethyl)benzene to form a monocyclic structure, and if you want to form a bicyclic structure, you must have three cysteines, and then react with 1,3,5-tris(bromomethyl)benzene and three mercaptogroups to form a bicyclic peptide.

[Solid-phase condensation of cyclic peptide-oligonucleotide conjugates, solid-phase ringing]
The above-mentioned method of solid-phase coupling liquid phase ring formation is used to prepare cyclic peptides, but this method still has certain shortcomings. We need to consider the stability of cysteine in ammonia, which can produce a large number of β elimination by-products if not paid attention to. The cyclization of peptides can be directly realized on the resin, on the resin is attached to a small C7-NH2 molecule, the N-terminal of this small molecule is protected by Fmoc, where peptide synthesis can be carried out, hydroxyl group is protected by DMTr, and solid-phase oligonucleotide synthesis can be carried out here. Then use 20% piperidine to remove the Fmoc protective group on the small molecule, and conjugate the amino acids in order, and then use a reducing agent such as DTT or dimercaptoethanol on the resin to remove the StBu protective group on the peptide cysteine first, so that the sulfhydride group on the peptide will leak out. The resin is then ring-forming with 1,3-dis(bromomethyl)benzene and exposed sulfhydryl groups. If the cyclization is successful, the solvent in the resin is drained, and then the solid-phase oligonucleotides are synthesized. Then the cyclic peptide-oligonucleotide conjugate is cut off from the resin with ammonia, and at this time, because the peptide has been successfully cyclocycized, the original cysteine has now become a sulfide bond, so there is no need to worry about the damage of ammonia to it, so as to avoid the elimination side reaction of the sulfhydryl group always encountered by the previous solid-phase synthesis of cyclic peptide-oligonucleotide conjugates.

Although linear cell transmembrane peptides can improve and enhance the delivery of oligonucleotide drugs, the presence of ubiquitous proteolytic enzymes makes linear cell-penetrating peptides unstable and limits the efficacy of oligonucleotides. Cyclization of peptides can improve their stability and improve oligonucleotide drug delivery. The current method of preparing cyclic peptide-oligonucleotide conjugates is mainly by liquid phase connection. However, cyclized cell transmembrane peptides usually contain multiple positively charged arginine or lysine, which will aggregate or precipitate with negatively charged oligonucleotides when connected by liquid phase method. The solid-phase method can avoid this problem of aggregation and precipitation.

Valprotide acetate is an artificially synthetic octapeptide somatostatin drug, which is clinically used for gastrointestinal bleeding, acromegaly, pancreatitis and other diseases, and also has a certain tumor inhibitory effect on neuroendocrine tumors, prostate cancer, etc. Therefore, using valprotide acetate as a template can improve the biocompatibility of precious metal nanomaterials. Valprotide acetate was approved for marketing in 2004 with the chemical name D-phenylalanyl-cysteine-tyrodyl-D-chromatoyl-lysazyl-valinel-cysteine-tryptamide-cyclo[2-7]-disulfide monoacetate.

Basic information:
Chinese name: valprotide acetate
English name: Vapreotide
Company number: GT-B016
CAS Number: 103222-11-3
Sequence: D-Phe-[Cys-Tyr-D-Trp-Lys-Val-Cys]-Trp-NH2
Formula: C57H70N12O9S2
Molecular weight: 1131.4

Physical and chemical properties
When valprotide acetate is dissolved in an acidic aqueous solution, it has a large positive charge on the surface because it is below its isoelectric point (pI≈10.0). In addition, because it is a small molecule compound composed of amino acids, the acidic environment will change the spatial conformation of the template, mainly manifested in α-helix and β-folding, etc., so that it can maintain a stable secondary and tertiary structure in aqueous solution for a long time. Similarly, when the valproptide acetate template is heat-treated at high temperature (≥70°C), its supersecondary structure (αα, βαβ, ββ) will change significantly, and the peptide chain forms a three-structure local fold area on the basis of the secondary structure or supersecondary structure, which is a relatively independent tight sphere, that is, the domain, which will also change due to changes in the environment. Of course, there are other means of processing peptide chains, such as heavy metal salts, ultrasound, etc. After the above acid and heat treatments, valprotide acetate will form a relatively stable three-dimensional structure, and the specific groups will be regularly exposed on the surface of the template. Therefore, when the metal salt solution is co-incubated with the pretreated Peptide acetate solution, the metal ions will bind to the surface of the template under the action of electrostatic forces and specific groups, and due to biomineralization, these attached ions will be reduced into trace small crystals, which we call "crystal seeds", and then under the action of suitable reducing agents, a large number of free metal ions are continuously reduced, and grow along one or several crystal planes of the "crystal seed", and finally form nanomaterials with the expected morphology.

Application
Vapapritide acid can be used as a biological template, and its advantages include: (1) small molecular weight, simple structure, and can be analyzed and designed according to the morphology of the intended material; (2) In acidic solution, the surface of vapritide acetate will carry a large amount of positive charge, and the precursor metal ions will be continuously adsorbed to the surface of the template through electrostatic force and specific groups, thereby increasing the metal load of nanomaterials and conducive to better their physical and chemical properties. (3) Valprotide acetate belongs to the somatostatin class, which has high biocompatibility, and the precious metal nanomaterials made from it as templates can be used in the field of physical medicine. Examples of its applications are as follows:
1) Preparation of a silver-gold alloy nanospherical shell based on valproptide acetate, It is mainly prepared according to the ratio of 0.91~1.13mg vapritide acetate per ml of hydrochloric acid solution, treated at 65~67°C for 20~24min, according to the ratio of 1:3~9, silver nitrate solution is added, put into a water bath constant temperature shaker at 24°C for 24~26h, according to the ratio of silver nitrate: sodium borohydride is 1:1~2, and then the reducing agent sodium borohydride is added drop by drop, and the reaction is 16~18min at 24°C; and then placed in a boiling water bath at 100°C for 10min, and gold trichloride solution is added according to the ratio of gold trichloride:silver nitrate molar ratio of 1:25~45 , 100°C boiling water bath for 2~4min, silver-gold alloy nanospheres with particle size of 90~110nm were prepared. The present invention is easy to control, simple to operate, green and environmentally friendly, mild reaction conditions, and the prepared silver-gold alloy has uniform particle size and good dispersion.
2) Preparation of palladium nanocubes. Valprotide acetate was dissolved in a hydrochloric acid solution with a pH of 1~3, A concentration of 0.1~0.2mM was prepared into 300μL vaprotide acetate solution, and the palladium chloride solution was added to the molar ratio of vaprotide acetate:palladium chloride at a ratio of 1:20~60, and placed in a constant temperature metal bath at 60~80°C. According to the molar ratio of palladium chloride:ascorbic acid was 1:10~50, the newly prepared ascorbic acid solution was quickly added for reduction, and the solution gradually changed from light yellow to black-brown, so as to obtain palladium nanocubes with regular morphology and uniform particle size. The preparation process of the present invention is simple and easy to operate; And this method does not use toxic reagents, which is green and environmentally friendly; The prepared palladium nanocubes have regular morphology, good photothermal conversion performance, and rapid heating after short-term low-power laser irradiation, which can be used as hyperthermic agents for cancer treatment.
3) Silver nanospheres were prepared with valprotide acetate as a biological template, It is mainly formulated into a vaprotide acetate solution with a concentration of 0.2~0.4mM, kept warm at 70~80°C for 10~30min, and the silver nitrate solution is added to the vapritide acetate solution at a ratio of 1:6~8, put into a water bath constant temperature shaker, 80~100rpm, 25°C for 12~24h, according to the molar ratio of silver nitrate solution: sodium borohydride is added to the incubated solution, the drop rate is 2 drops/minute, 60μL per drop, 25°C reaction for 5~15min, so as to obtain silver nanospherical shells with uniform particle size and good dispersion of 80~100nm. The silver nanosphere shell prepared by the invention overcomes the shortcomings of easy aggregation and precipitation in the traditional preparation process, and has a high metal load.

Anyone need customized peptides, plz contact haoran.tse@gmail.com
Telphone no: +8618575536586

1. Introduction

Peptides are compounds formed by two or more amino acids linked by peptide bonds, which are widely present in living organisms and participate in a variety of physiological processes, including cell signaling, enzyme catalysis, immune response, and metabolic regulation. In recent years, with the development of biosynthesis and chemical synthesis technologies, peptide customization services have increasingly become a core tool in life sciences, pharmaceutical research and development, and molecular biology research.
The editor will introduce in detail the technical principles, main processes, quality control processes and typical applications of peptide customization in scientific research.

2. Technical principle of peptide customization

Peptide customization is the process of synthesizing amino acids in specific sequences to obtain the target peptide molecules with the desired structure and function. Its main synthesis techniques include:

1. Solid Phase Peptide Synthesis (SPPS)
   At present, a wide range of synthesis technologies are used. By fixing one amino acid on a solid-phase resin, then gradually adding other amino acids, using condensing agents (e.g., HBTU, DIC) to connect the amino acid residues, and finally chemically cutting the synthesized peptides from the solid-phase carrier.
   Advantages: high degree of automation; can synthesize long peptides; Easy to purify and modify.
2. Liquid Phase Synthesis
   It is suitable for short-chain peptides and large-scale industrial synthesis, with mild reaction conditions but cumbersome steps.

3.Detailed explanation of the customization process

A standard peptide customization process generally includes the following key steps:

1. Design and sequence confirmation
   Customers provide amino acid sequence or functional requirements;
   The service party analyzes the sequence (whether it contains special modifications, whether it contains disulfide bonds, whether it is water-soluble, etc.);
   Bioinformatics tools were used to predict secondary structure, hydrophilicity and other parameters to assist in design.
2. Synthesis stage
   Choose the appropriate solid-phase carrier (e.g., Rink amide resin, Wang resin);
   Precise control of the coupling reaction of each amino acid;
   Add protective groups (e.g., Fmoc, Boc) to avoid side effects.
3. Purification and analysis
   High Performance Liquid Chromatography (HPLC) for separation and purification, the purity can reach more than 95%;
   Molecular weight was confirmed using mass spectrometry (MALDI-TOF, ESI-MS);
   Nuclear magnetic resonance (NMR) and circular dichroism (CD) assisted analysis are performed if necessary.
4. Packaging and quality control
   Provide lyophilized powder according to customer needs;
   Evaluation of storage conditions by stability tests (high temperature, light, hydrolysis);
   Provide Quality Analysis Report (COA) and MSDS documentation.

4. Types of peptide modifications

Common functional touch-ups in the customization process include:
N-terminal modifications: acetylation, biotin, biotinylation;
C-terminal modification: amideation, esterification;
Fluorescent labeling: FITC, TAMRA;
phosphorylation/glycosylation: mimicking naturally modified peptides;
Disulfide bond bridging: stabilize the spatial structure.

5. Application of peptide customization in scientific research

1. Antibody preparation and epitope analysis
   Researchers can synthesize specific epitope peptides as antigens for the preparation of polyclonal or monoclonal antibodies.
2. Cell signaling pathway research
   Synthesis of phosphorylated or mutant site peptides for protein kinase activity screening, pathway validation, etc.
3. Pharmacological screening and functional verification
   peptide antagonists and agonist mimics;
   Combination studies with GPCR and other targets.
4. Immunology research
   Vaccine research and development, immunogenicity assessment, peptide library construction, etc.
5. Protein interaction experiments (e.g., pull-down)
   Protein function and interaction analysis are achieved by capturing protein complexes with tags (e.g., His, FLAG, Biotin) with peptides.

6. Technical challenges and development trends and Challenge:

Low synthesis efficiency of long-chain (>50aa) peptides;
Hydrophobic polypeptides are easy to aggregate and affect solubility;
The synthesis steps of special modifications (such as glycosylation) are complex and costly.
Trend:
Accelerate the promotion of intelligent and peptide synthesis automation equipment;
Peptide drug development drives breakthroughs in functional modification technology;
Custom processes are integrated with bioinformatics to improve design accuracy.

7. Summary

Peptide customization is a highly integrated biosynthesis and chemical engineering technology that requires not only precise equipment and materials, but also precise design logic and analytical capabilities. In the field of scientific research, it has become an indispensable technical support for experimental design and biological validation. With the upgrading of technology and the deepening of application, it will release greater potential in the future in basic scientific research, precision medicine, biopharmaceuticals and other directions.