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引言
氨基酸是蛋白质的基本合成子，是生命的重要组成部分。它们有相同的主链结构，包括一个碱性氨基（-NH2）、一个酸性羧基（-COOH）和一个特有的有机R基（或侧链）组成，如式1。
式1 氨基酸
自然界中有多种氨基酸，如编码蛋白质的20种常见氨基酸。而非天然氨基酸（unnatural amino acid, UAA）则是指生物体中非天然存在或不参与蛋白质合成的氨基酸，作为天然氨基酸的衍生物，他们有着相同的骨架结构，且带有结构多样的侧链基团，从而具有独特的化学性质，包括生物催化活性、结构和热学稳定性及底物特异性等，这些特性的改变使其在生命科学、医药、农业等多个行业有着巨大的应用潜力。
尽管UAA有着巨大的应用潜力，但其合成仍是一个挑战。虽然有一系列的化学合成方法用于制备UAA，但传统有机合成法通常依赖高温高压条件、贵金属催化剂及繁琐的保护/脱保护步骤，不仅导致产物收率与光学纯度受限，还产生大量环境污染物，难以满足绿色制造的产业需求。酶催化技术作为绿色合成的核心手段，具有反应条件温和（常温常压、水溶液体系）、立体选择性高（对映体过量值e.e.可达95%-99%）、原子经济性好等优势，已成为UAA合成的研究热点。
非天然氨基酸的酶催化合成路线
为满足市场对更加绿色，更加经济的合成非天然氨基酸的需求，博腾生物催化团队开发了非天然氨基酸的生物催化合成平台，见式2，并孵育了数十个规模从公斤级到吨级不等的项目。主要包括以下四种技术路径：
式2 博腾非天然氨基酸生物催化合成平台
① 生物催化酮酸合成非天然氨基酸
从潜手性酮酸出发，生物催化合成非天然氨基酸的策略有两种：酶催化酮酸的不对称还原胺化，和酶催化酮酸的不对称氨基转移反应，见式3。
式3 生物催化酮酸合成非天然氨基酸
酶催化酮酸的不对称还原胺化是生产手性氨基酸的高效策略之一。该反应由氨基酸脱氢酶（AADH），如谷氨酸脱氢酶（GluDH）、亮氨酸脱氢酶（LeuDH）、苯丙氨酸脱氢酶（PheDH）等，在NH4?存在下，以NAD(P)H为辅因子进行可逆催化（式3A）。昂贵辅酶（NADH和NADPH）的循环再生是该技术工业化应用的关键。甲酸脱氢酶（FDH）和葡萄糖脱氢酶（GDH）可用于该类辅酶的循环再生。另外，该策略可分别用不同选择性的AADHs合成D-型和L-型氨基酸。
酶催化酮酸的不对称转氨反应是另一种从酮酸出发合成非天然氨基酸的高效策略。该反应由α-氨基转移酶（α-ATA），在5'-磷酸吡哆醛（PLP）的存在下，催化氨基供体的氨基转移至氨基受体酮酸的羰基碳原子上（式3B）。该反应是可逆的，常常面临反应平衡问题。投入过量氨基供体，和/或通过其他酶促反应移除副产物可推动反应向目标方向进行。
在一个芳香族氨基酸的合成项目中，见式4，原工艺路线从芳香醛（SM1）出发，总计7步化学反应（含2步不分离步骤）。采用昂贵的铑（Rh）催化剂和手性配体催化不对称氢化的方式来构建手性中心，且需要在~5 atm的氢气下进行，中间体3的e.e%仅为94.4%，需要进一步结晶纯化到e.e%>99.0%。再经脱乙酰基，脱甲基，成盐，上Fmoc保护得到目标产品。路线的收率仅为38%，成本极高，不适合工业化生产。博腾生物催化团队随即开发了化学-酶催化路线。该路线同样从SM1出发经中间体1（不分离）直接合成中间体7（α-酮酸），然后经酶催化（2种不同的方法：α-氨基转移酶介导的氨基转移和氨基酸脱氢酶/甲酸脱氢酶介导的还原胺化）得到光学纯的游离氨基酸8，该手性构建步骤反应温和（常压，25℃，pH8-9），得到游离氨基酸8光学纯度e.e%>99.0%，化学纯度高，不需要进一步纯化，简单的分离即可上Fmoc保护得到目标产品。该路线总反应步骤较原路线减少至4步（含1步不分离步骤），总收率提高至57.5%。成本较原路线降低>50%，已经成功吨级交付并持续交付中。
式4 芳香族氨基酸合成路线，化学法 VS 化学-酶法
作为平台技术，该路线还测试了一系列的底物，见表1。均可利用该路线得到高光学纯度的D-或L-型目标氨基酸，且有多个已经实现公斤级-吨级交付。
表1
② 生物催化α,β-不饱和羧酸合成非天然氨基酸
氨裂解酶（如苯丙氨酸氨裂解酶PALs、甲基天冬氨酸氨裂解酶MALs、天冬氨酸氨裂解酶DALs等）催化α,β-不饱和羧酸双键和NH4+的对映选择性加成，是生产手性氨基酸的另一种高效策略（式5）。该反应无需任何的辅因子、原料α,β-不饱和羧酸易于合成且原子经济性非常好。由于该转化反应同样可逆，仍需过量NH4?推动反应向目标方向进行。
式5 生物催化α,β-不饱和羧酸合成非天然氨基酸
在一个杂环芳香族氨基酸的合成项目中，原化学路线同样采用手性源和贵金属不对称氢化的方式构建手性中心，成本高，过程冗长，手性纯度较低，需要进一步纯化。博腾生物催化团队则开发了另一条化学-酶催化路线，如式6。
式6 化学-酶催化法合成杂环芳香族氨基酸
该路线从杂环芳香醛(RM1)出发，经与丙二酸缩合得到中间体9（烯酸），再经氨基酸裂合酶催化得到光学纯的游离氨基酸10，最后上Boc保护得到目标产品，光学纯度高达99.8%。整条路线仅3步，较化学路线大幅减少。总成本较原路线降低>90%。已经成功吨级交付并持续交付中。
同样，该路线还测试了一系列的底物，见表2。均可利用该路线得到高光学纯度的D-或L-型目标氨基酸，且有多个已经实现公斤级-吨级交付。
表2
③ 生物催化合成色氨酸衍生物
酶直接催化吲哚衍类似物和丝氨酸（或丙酮酸与NH4?）缩合，是制备色氨酸衍生物的最直接且高效的策略（式7）。以5'-磷酸吡哆醛（PLP）为辅酶，色氨酸合成酶（TrpS）和色氨酸酶（Tryptophanase）可催化吲哚衍生物与丝氨酸（Ser）形成C-C键，生成L-色氨酸（Trp）衍生物。另外色氨酸酶还能以丙酮酸和NH4?替代丝氨酸作为底物。
式7 酶催化合成色氨酸衍生物
消旋的乙酰氨基酸11，然后再经酰基水解酶水解拆分，将S-型水解成目标构型12，而将R-型原料11’完全保留，经分离后丢弃。中间体12经分离再上Boc保护得到目标产品。该路线是拆分工艺，中间体11的一半无法转化为目标产品，被直接丢弃，整体收率仅19.7%，原子经济性极差。博腾生物催化团队开发了一条化学-酶催化路线，该路线同样从取代吲哚RM1出发，直接利用色氨酸合成酶/色氨酸酶催化与L-丝氨酸或丙酮酸加铵根离子缩合，得到光学纯度>99%的氨基酸12，不需分离纯化即可直接上Boc保护得到目标产品。路线仅2步，且可一锅进行。收率高达90%，总成本较原路线降低>90%。已经成功吨级交付并持续交付中。
式8 化学-酶催化法合成色氨酸衍生物
同样，该路线还测试了一系列的取代吲哚底物，见表3。均可利用该路线得到高光学纯度的目标氨基酸，且有多个已经实现公斤级-吨级交付。
表3
④ 多酶级联催化合成非天然氨基酸
虽然已经开发了多条包含酶催化的合成路线合成各类非天然氨基酸，但依然面临多步反应，多次分离、提取、过滤、干燥等耗时工序。而利用一锅多步酶法级联的方式则可有效解决上述问题。如式9，该路线从醛出发，利用苏氨酸醛缩酶催化与甘氨酸缩合生成中间体13，再经苏氨酸脱水酶催化得到中间体14，再经氨基酸脱氢酶/甲酸脱氢酶体系催化得到高光学纯度的游离氨基酸15，不用分离可直接上Boc保护得到目标产品。该路线随经多步酶催化反应，但采用一锅法，不用分离任何一个中间体直接一步做到目标产品，总收率可达65-70%，较分步工艺提高10+%。且整个生产周期缩短>50%，生产成本大幅度降低。
式9 一锅4酶级联催化合成非天然氨基酸
同样，该路线还测试了一系列的底物，见表4。均可利用该路线得到高光学纯度的D-或L-型目标氨基酸。
表4
技术瓶颈与未来布局
尽管生物催化合成UAAs技术发展迅速，行业整体仍面临若干共性技术瓶颈：
酶的底物专一性限制了一些底物难以通过现有酶催化体系合成。
酶的稳定性较差，活性易受反应条件影响而降低，甚至失活。
多酶级联路线中各酶的加入量需要精确调控以避免中间产物积累，减轻由此导致的抑制和毒害的问题。
针对上述挑战，博腾生物催化技术平台将持续深耕生物催化合成非天然氨基酸领域，重点聚焦以下五大研发方向：
新路线、新酶的持续开发，现有路线酶的再开发，开发具有底物广谱性、高活性、高稳定性的新型酶催化剂。
扩展不同类型UAAs的化学-酶、生物合成技术。
模块化设计，构建多种多酶级联的生物催化合成路线。
发展酶的固定化、全细胞催化技术、微生物发酵与生物合成协同工艺，降低生产成本，推动工业化应用。
加强跨学科合作，结合化学合成、流体化学、光化学等先进技术开发更多的UAAs合成的平台技术。
结语
生物催化技术正逐步成为非天然氨基酸绿色合成的核心驱动力。博腾生物催化技术平台通过持续开发新酶、优化多酶级联体系、拓展化学-酶协同路线，已在UAAs合成领域建立了扎实的技术积累。针对底物专一性、酶稳定性与工业化放大等行业共性挑战，博腾将持续聚焦新型酶催化剂开发与跨学科技术融合，致力于缩短生物催化技术从实验室研究到工业化应用的周期，为客户提供更具成本竞争力与环境友好性的非天然氨基酸合成解决方案。
关于博腾生物催化技术平台
博腾生物催化技术平台依托经验丰富的专职团队和先进设备，专注于为客户提供包括生物催化开发、酶制备和生物催化生产在内的“一站式”、“端到端”CDMO服务。平台建有覆盖数十种反应类型的自有酶库，并配套高通量筛选与数据驱动的新酶发现体系。平台还具备从实验室研发至中试规模的发酵能力与酶固定化技术，拥有丰富的放大生产经验。博腾通过优化反应条件与工艺路径，在提升合成效率的同时，积极践行绿色制药理念，致力于让好药更早惠及大众。
博腾股份成立于2005年，为全球药企、生物科技公司及科研机构等提供从临床前研究到药品上市全生命周期的端到端CDMO服务，涵盖小分子药物、多肽与寡核苷酸药物、蛋白与偶联药物以及细胞与基因治疗药物的原料药到制剂全流程。截至2025年末，公司拥有全球员工4300余人，累计服务客户1400余家，成功交付项目超5000个。我们始终坚持以客户为中心，致力于提供创新可靠的全球化CDMO解决方案，让好药更早惠及大众。
Introduction
Amino acids are the fundamental building blocks of proteins and essential components of life. They share a common backbone structure consisting of a basic amino group (-NH2), an acidic carboxyl group (-COOH), and a unique organic R-group (or side chain), as shown in Scheme 1.
Scheme 1. Amino Acid Structure
Numerous amino acids have been discovered in nature, including the 20 canonical amino acids that encode proteins. In contrast, unnatural amino acids (UAAs) refer to amino acids that do not naturally exist in organisms or do not participate in protein synthesis. As derivatives of natural amino acids, they retain the same backbone structure and feature structurally diverse side chains, endowing them with unique chemical properties such as biocatalytic activity, enhanced structural and thermal stability, and substrate specificity. These modified properties make UAAs highly valuable for applications in life sciences, medicine, and agriculture.
Despite their immense commercial potential, the synthesis of UAAs remains challenging. Although various chemical synthesis methods exist, traditional organic synthesis often relies on harsh conditions (high temperature/pressure), expensive metal catalysts, and tedious protection/deprotection steps. These limitations result in low yields, poor optical purity, and significant environmental pollution, failing to meet the demands of green manufacturing.
Enzyme-catalyzed synthesis, as a core strategy for green chemistry, offers mild reaction conditions (ambient temperature/pressure, aqueous systems), high stereoselectivity (enantiomeric excess, e.e., up to 95%-99%), and excellent atom economy. Consequently, it has emerged as a key research focus for UAA production.
Enzymatic Synthesis Routes for Unnatural Amino Acids
To meet the market demand for greener and more cost-effective approaches forUAA synthesis, Porton Biocatalysis team has developed an enzymatic synthesis platform for UAAs (see Scheme 2) and delivered dozens of projects at scales ranging from kgs to tons, including the following four main approaches:
Scheme 2. Enzymatic Synthesis Platform for UAAs at Porton
① Biocatalytic Synthesis of UAAs from Keto-acids
There are two enzymatic UAA synthesis strategies starting from prochiral keto-acids: enzymatic asymmetric reductive amination of keto-acids and asymmetric transfer of amino group to keto-acids, see Scheme 3.
Scheme 3. Enzymatic Synthesis of UAAs from Keto-acids
Enzymatic asymmetric reductive amination of keto-acids is a very efficient strategy for the production of chiral amino acids. The reaction is catalyzed reversibly by amino acid dehydrogenase (AADH), like glutamate dehydrogenase (GluDH), leucine dehydrogenase (LeuDH), phenylalanine dehydrogenase (PheDH), etc., using NAD(P)H as cofactor and at the presence of NH4+(Scheme 3A). Efficient regeneration of the costly cofactors NADH and NADPH is critical for the industrial application of this strategy. Formate dehydrogenase (FDH) and glucose dehydrogenase (GDH) are most frequently used to regenerate these cofactors. In addition, this strategy enables the synthesis of both D- and L-isomers of amino acids through the use of AADHs with different stereoselectivities.
Enzymatic asymmetric transamination is another efficient strategy for synthesizing UAAs from keto acids. (Scheme 3B). The reaction is catalyzed reversibly by α-aminotransferase (α-ATA), which transfer of an amino group from amino donor to the carbonyl carbon of keto-acid as amino acceptor at the presence of pyridoxal 5’-phosphate (PLP). This reaction is reversible and therefore equilibrium often becomes an issue. To drive the conversion toward the desired direction, more than stoichiometric amounts of amino donor are charged and/or the by-product is removed by other enzymes.
In a project of aromatic amino acid synthesis (see Scheme 4), the original process is a 7-steps (including 2 non-isolation steps) process starting from the corresponding aromatic aldehyde (SM1), the chiral center was constructed through asymmetric hydrogenation by expensive Rh-catalyzed and chiral ligands at ～5 atm hydrogen. Affording intermediate 3 with 94.4% in e.e.%, necessitating further crystallization to achieve >99.0% in e.e.%. The target product can be finally obtained through subsequent steps including deacetylation, demethylation, salt formation, and Fmoc protection. The overall yield of this route was merely 38%, resulting in a prohibitively high costs and an unsuitable approach for commercial production.
Porton Biocatalysis team subsequently developed an alternative chem-enzymatic route. Started from the same SM1 to synthesize intermediate 1 and proceed to intermediate 7 (keto-acid) directly without isolation and purification. The chiral center was then established via enzymatic catalysis (two approaches: α-transaminase-mediated transamination or amino acid dehydrogenase/formate dehydrogenase-mediated reductive amination), yielding optically pure free amino acid 8. This chiral construction step run under mild conditions (ambient pressure, 25°C, pH 8–9), achieving >99.0% e.e.% without further purification. Then, the target product can be obtained through Fmoc protection with simple workup of free amino acid 8.
In the alternative chem-enzymatic route, the synthesis sequence was reduced to 4 steps (including 1 non-isolation step), the overall yield was increased to 57.5% and the overall cost was lowered by >50% compared to the original process. It has been successfully implemented at ton-scale and remains in continuous delivery.
Scheme 4. Synthesis of Aromatic Amino Acid, Chemical Route vs Chem-enzymatic Route
As a platform technology, this approach has been tested with a series of substrates (see Table 1). The corresponding amino acids, both D- and/or L-enantiomer can be obtained with high optical purity, and several of them have already been delivered at kg-to-ton scale.
Table 1
② Biocatalytic Synthesis of UAAs from α, β-Unsaturated Carboxylic Acids
Ammonia lyases, such as phenylalanine ammonia lyases (PALs), methylaspartate ammonia lyases (MALs) and aspartate ammonia lyases (DALs), etc., catalyze enantioselective addition of ammonia to double bonds of α, β-unsaturated carboxylic acids is an attractive strategy for the production of chiral amino acids (Scheme 5). In this strategy, no cofactor is needed, α, β-unsaturated carboxylic acids can be synthesized easily and the atom economy is perfect. Since this conversion is also reversible, excess NH4+ is also needed to push forward to the desired direction.
Scheme 5. Enzymatic Synthesis of UAAs from α, β-Unsaturated Carboxylic Acids
In a project of heterocyclic aromatic amino acid synthesis, chiral pool and transition metal-catalyzed asymmetric hydrogenation were employed to construct the chiral center in the original chemical route, which was costly, lengthy, and yielded low chiral purity and further purification was needed. An alternative chemoenzymatic route developed by biocatalysis team, as shown in Scheme 6.
Scheme 6. Synthesis of Heterocyclic Aromatic Amino Acid by Chemical-enzymatic Route
In the alternative route, intermediate 9 (unsaturated acid) was obtained by condensation of the corresponding heterocyclic aromatic aldehyde and malonic acid. Then catalyzed by an amino acid lyase to produce optically pure free amino acid 10, which was then protected by Boc anhydride to obtain the target product with an optical purity of up to 99.8%. It was a 3 steps process, which was significantly shorter than the chemical route and the total cost is reduced significantly by >90%. It has been successfully implemented at ton-scale and remains in continuous delivery.
Similarly, a series of substrates has been tested via this approach (see Table 2). The corresponding amino acids, both D- and/or L-enantiomer can be obtained with high optical purity, and several of them have already been delivered at kgs scale.
Table 2
③ Biocatalytic Synthesis of UAAs from Keto-acidsBiocatalytic Synthesis of Tryptophan Derivatives
Direct enzymatic alkylation of indole derivatives with serine and/or pyruvic acid and NH4+ is a direct and potentially efficient approach in the preparation of a number of tryptophan derivatives (Scheme 7). Tryptophan synthase (TrpS) and tryptophanase, two PLP pyridoxal 5’-phosphate (PLP)-dependent enzymes, catalyze the C–C bond forming reaction between indole derivatives and serine (Ser) to make L-tryptophan (Trp) derivatives, and tryptophanase can also accept pyruvic acid and NH4+ replacing serine.
Scheme 7. Enzymatic Synthesis of Tryptophan Derivatives
In a project of tryptophan derivative synthesis (see Scheme 8), racemic amino acid was obtained through condensation of substituted indole RM1 and L-serine in the original route, which was then acetylated to yield racemic intermediate 11. The target isomer was hydrolyzed by acylase to give the desired enantiomer 12 while leaving the R-isomer 11' not been hydrolyzed, which was discarded after separation. The final product was obtained by Boc-protection of intermediate 12 after isolation. As a resolution process, half of intermediate 11 could not be converted into the target product and was directly discarded, resulting in an overall yield of only 19.7% and extremely poor atom economy.
To overcome these drawbacks of resolution process, an alternative chemoenzymatic route was developed. From the same starting materials, amino acid 12 can be obtained with >99% optical purity by tryptophan synthase/tryptophanase catalyzed condensation of the substituted indole RM1 and L-serine or pyruvate/NH4+. Without further purification and it can be proceeded to the target compound directly by Boc-protection. It is a one-pot 2 steps process which achieves a remarkable yield of 90% and reduces total costs by >90% compared to the original process. This process has been successfully implemented at ton-scale and remains in continuous delivery.
Scheme 8. Synthesis of Tryptophan Derivatives by Chemical-enzymatic Route
A series of substituted indole derivatives were tested (see Table 3). The corresponding tryptophan derivatives can be obtained with high optical purity, and several of them have already been delivered at kgs scale.
Table 3
④Synthesis of UAAs via Multi-enzyme Cascades System
Although multiple synthetic routes incorporating enzymatic catalysis have been developed for various UAAs, which still require multi steps and time-consuming operations such as repeated isolation, extraction, filtration, and drying.
The use of a one-pot, multi-step enzymatic cascade process can effectively address these challenges. As shown in Scheme 9, threonine aldolase is introduced to catalyze the condensation of aldehyde and glycine to give intermediate 13 in this approach, which is subsequently converted to intermediate 14 by threonine dehydratase. Then an amino acid dehydrogenase/formate dehydrogenase system is introduced to give free amino acid 15 with high optical purity. At last, the target product can be obtained after Boc-protection.
As no isolation step needed, the total yield reaches 65–70%, representing a >10% improvement over stepwise processes. Additionally, the production cycle is shortened by >50%, and manufacturing costs are significantly reduced.
Scheme 9. Synthesis of UAAs by One-pot 4-enzyme Cascades
A series of substrates were tested via this approach (see Table 4). The corresponding amino acids, both D- and/or L-enantiomer can be obtained with high optical purity, and several of them have already been delivered at kgs scale.
Table 4
Remaining Challenges and Path Forward
Despite rapid progress in biocatalytic UAA synthesis, several industry-wide
Due to the specificity of enzyme, some substrates are difficult to synthesize using existing enzyme systems.
Enzymes are prone to inactivation under certain reaction conditions.
Precise control of enzyme (activity) loading is required to prevent intermediate accumulation, which may lead to inhibition and toxicity.
To tackle these challenges, Porton Biocatalysis Technology Platform will focus on the following key areas to enable more cost-effective and efficient UAA synthesis:
Develop new enzymes with broad substrate spectrum, high activity, and enhanced stability.
Develop diverse chemoenzymatic and biosynthetic strategies for various UAAs.
Modular design for multi-enzyme cascades processes.
Develop enzyme immobilization, whole-cell catalysis, etc., to lower production costs and enable industrial-scale applications.
Integrating chemical synthesis, flow chemistry, photochemistry, and other advanced technologies to establish versatile UAA synthesis platform.
Conclusion
Biocatalysis is a pivotal enabler for the sustainable synthesis of unnatural amino acids. By integrating novel enzymes discovery, multi-enzyme cascades and chemo-enzymatic routes, Porton has established practical capabilities to address industry challenges in substrate specificity and enzyme stability. Moving forward, Porton remains dedicated to bridging the gap between laboratory innovation and industrial-scale application, providing customers with reliable, scalable and atom-efficient solutions for UAA production.
Founded in 2005, Porton Pharma Solutions provides end-to-end CDMO services from pre-clinical to commercial stages. Our capabilities span Small Molecules, TIDES, Biologics and Conjugates (ADCs, AOCs, DACs, PDCs, RDCs, etc.), and Advanced Therapy Medicinal Products, from drug substance to drug product. As of 2025, Porton has over 4,300 employees globally, served more than 1,400 customers, and delivered over 5,000 projects. We are committed to a customer-centric approach, delivering innovative and reliable global CDMO solutions, enabling earlier access to good medicines.
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