Background: Protein pharmaceuticals routinely display a series of intrinsic physicochemical instabilities during their production and administration that can unfavorably affect their therapeutic effectiveness. Glycoengineering is one of the most desirable techniques to improve the attributes of therapeutic proteins. One aspect of glycoengineering is the rational manipulation of the peptide backbone to introduce new N-glycosylation consensus sequences (Asn-X-Ser/Thr, where X is any amino acid except proline).

Methods: In this work, the amino acid sequence of human chorionic gonadotropin (hCG) was analyzed to identify suitable positions in order to create new N-glycosylation sites. This survey led to the detection of 46 potential N-glycosylation sites. The N-glycosylation probability of all the potential positions was measured with the NetNGlyc 1.0 server. After theoretical reviews and the removal of unsuitable positions, the five acceptable ones were selected for more analyses. Then, threedimensional (3D) structures of the selected analogs were generated and evaluated by SPDBV software. The molecular stability and flexibility profile of five designed analogs were examined using Molecular Dynamics (MD) simulations.

Results: Finally, three analogs with one additional N-glycosylation site (V68T, V79N and R67N) were proposed as the qualified analogs that could be glycosylated at the new sites.

Conclusion: According to the results of this study, further experimental investigations could be guided on the three analogs. Therefore, our computational strategy can be a valuable method due to the reduction in the number of the expensive, tiresome and time-consuming experimental studies of hCG analogs.

背景：蛋白药物在生产和给药过程中通常会表现出一系列内在的物理化学不稳定性，这会不利地影响其治疗效果。糖工程是改善治疗性蛋白质的最理想的技术之一。糖工程的一个方面是合理控制肽主链以引入新的N-糖基化共有序列（Asn-X-Ser / Thr，其中X是脯氨酸以外的任何氨基酸）。

方法：在这项工作中，分析了人类绒毛膜促性腺激素（hCG）的氨基酸序列，以确定合适的位置，以创建新的N-糖基化位点。这项调查导致发现了46个潜在的N-糖基化位点。使用NetNGlyc 1.0服务器测量了所有潜在位置的N-糖基化概率。经过理论审查并排除了不合适的职位之后，选择了五个可接受的职位进行更多分析。然后，通过SPDBV软件生成并评估所选类似物的三维（3D）结构。使用分子动力学（MD）模拟检查了五个设计类似物的分子稳定性和柔韧性。

结果：最后，提出了三个具有一个额外的N-糖基化位点的类似物（V68T，V79N和R67N），作为可以在新位点进行糖基化的合格类似物。

结论：根据这项研究的结果，可以对这三种类似物进行进一步的实验研究。因此，由于减少了hCG类似物的昂贵，繁琐和耗时的实验研究，我们的计算策略可以成为一种有价值的方法。