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The macrocycle 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is an excellent, versatile chelator of metals. Previously published research has shown that DOTA is the preferred ligand for trivalent yttrium and lanthanide ions. However, other radiometals can be used with DOTA. Over the past 30 years, DOTA has become the dominant chelator used in PET and SPECT imaging and in radiotherapy. Quanta BioDesign offers many of our DOTA-dPEG® product lines in both DOTA-tris(TBE) and DOTA-tris(acid) forms of the products. See Figure 1, below, for an example. Consequently, some customers ask us, “Which form is right for my application?” The answer we give depends upon the type of application with which a particular customer is working.

Figure 1: Comparison of PN11155, DOTA-tris(TBE)-amido-dPEG®4-TFP ester, with PN11160, DOTA-tris(acid)-amido-dPEG®4-TFP ester
Figure 1: Comparison of PN11155, DOTA-tris(TBE)-amido-dPEG®4-TFP ester, with PN11160, DOTA-tris(acid)-amido-dPEG®4-TFP ester


The tris(TBE) form (“TBE” stands for “tert-butyl ester”) of our DOTA products is a protected intermediate to the tris(acid) form of the product. In this form, the tert-butyl ester groups protect the three remaining acetate groups on the DOTA. For certain applications, unprotected acetate groups may react in subsequent manipulation steps.

Removal of tert-butyl ester groups from DOTA-tris(TBE)

The tert-butyl ester groups are removed using 2,2′,2”-trifluoroacetic acid (TFA). In our hands, approximately 60 molar equivalents of TFA relative to the DOTA-tris(TBE)-dPEG® product are required for complete removal of the tert-butyl ester groups. Importantly, the reaction may require up to 72 hours at ambient temperature to complete. Following removal of the tert-butyl ester groups, the acetate moieties are free to bind metal radionuclides.

Caveat Regarding TFA

Generally, we caution our customers that it may be impossible to remove all of the TFA from the final product. Indeed, we sell our DOTA-tris(acid) products as TFA salts, because of the difficulty in removing TFA. Please see the protocol at the end of this article for possible ways to remove most of the TFA from the deprotection reaction.

How to decide whether to use DOTA-tris(TBE) or DOTA-tris(acid)

The user must understand the molecule to which the DOTA-dPEG® functionality is to be added when deciding whether to conjugate the protected (TBE) or unprotected (acid) form of our DOTA products to a molecule. Reaction chemistry and molecular stability guide the user in selecting which DOTA product to add and in deciding when to add it. Critically, if the bioconjugate product cannot withstand the use of large amounts of TFA for an extended period of time in the deprotection reaction, then the acid form of the DOTA product should be used.

When a customer wants to incorporate a DOTA product into a molecule (small molecule or peptide, for example) at the end of the synthetic process, then we recommend that the use of a DOTA-tris(acid) product, because it does not require deprotection of the TBE groups. In contrast, when further reaction steps follow the insertion of the DOTA functional group, we strongly recommend that the customer consider the subsequent reaction steps before deciding what DOTA product to use.

First, if the subsequent reaction steps are compatible with free acids, then you may use either tris(TBE) or tris(acid) products. Otherwise, use DOTA-tris(TBE) products.

Second, if the final product is compatible with TFA deprotection of the DOTA-tris(TBE) group to form the tris(acid) product, then use a DOTA-tris(TBE) product. If this is not the case, then use a DOTA-tris(acid) product.

Third, if subsequent reaction steps are not compatible with free acids (the first consideration), and if the final product is not compatible with TFA deprotection of the TBE (the second consideration), then we recommend that the user redesign the synthetic process so that the DOTA-tris(acid) product is added as the last step in the process.

Why use a DOTA-dPEG® reagent instead of just DOTA?

Our DOTA-dPEG® products offer three principal advantages over DOTA alone. First, dPEG® products increase the hydrodynamic volume of molecules to which they are conjugated. Increased hydrodynamic volume reduces renal excretion in vivo. Consequently, this leads to prolonged circulation time. Second, dPEG® products are non-immunogenic and have been shown to reduce the immunogenicity of conjugates made with dPEG® products. Third, in 2004, Rogers, Della Manna, and Safavy reported that a DOTA-PEG conjugate (using disperse PEG with Mw of 3,500) had a better biodistribution profile than a bioconjugate without PEG. Subsequently, other conjugates demonstrated improved performance with DOTA-PEG compared to DOTA alone (see references 7, 8, and 9, below). For other advantages of dPEG® over disperse PEG, please see our frequently asked questions section, particularly here and here.

Protocol for Deprotecting DOTA-tris(TBE) with TFA

This protocol outlines the use of TFA to remove tert-butyl ester groups from DOTA-tris(TBE). This protocol proceeds from the premise that DOTA-tris(TBE) has been conjugated to a molecule of interest (e.g., a peptide). Thus, deprotection using TFA is now the final step to be accomplished.

  1. Dissolve a known amount of DOTA-tris(TBE)-dPEG® conjugate in dichloromethane or another suitable solvent.
  2. Pour the conjugate solution into a suitable reaction vessel.
  3. Place the reaction vessel in an ice bath and chill with stirring.
  4. By drop, add 60 molar equivalents of TFA into the reaction vessel with stirring.
  5. After all of the TFA has been added, remove the reaction vessel from the ice bath and allow the reaction to come to room temperature.
  6. Monitor the reaction progress by RP-HPLC or TLC until it is complete. The deprotection process is slow. Some reactions may require as long as 72 hours to complete.
  7. Once the reaction is complete, remove the solvent by rotary evaporation or any other suitable method.
  8. Remove the TFA from the product either by chromatography or by triturating the product at least four (4) times with ice cold diethyl ether. Please note that some TFA may remain bound to the DOTA moiety after this treatment.


We are always happy to receive technical questions from our customers on how to use our products to greatest effect. We strive to help you succeed with our products! When you have questions, please feel free to contact us.


DOTA Products That May Interest You

11155, DOTA-tris(TBE)-amido-dPEG®4-TFP ester 11160, DOTA-tris(acid)-amido-dPEG®4-TFP ester
11157, DOTA-tris(TBE)-amido-dPEG®12-TFP ester 11162, DOTA-tris(acid)-amido-dPEG®12-TFP ester
11158, DOTA-tris(TBE)-amido-dPEG®24-TFP ester 11163, DOTA-tris(acid)-amido-dPEG®24-TFP ester


11166, DOTA-tris(acid)-amido-dPEG®11-Maleimide 11167, DOTA-tris(acid)-amido-dPEG®11-Maleimide
11171, DOTA-tris(acid)-amido-dPEG®23-Maleimide  



  1. Hermanson, G. T. Chapter 2, Functional Targets for Bioconjugation. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 127-228. Most bioconjugation experts consider Greg Hermanson’s book to be one of the best references for this field. Please click here for a review of this book and to purchase it.
  2. Hermanson, G. T. Chapter 12, Isotopic Labeling Techniques. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 507-534, specifically pages 508-509, discussing DOTA.
  3. Hermanson, G. T. Chapter 18, PEGylation and Synthetic Polymer Modification. Bioconjugate Techniques, 3rd edition. Academic Press: New York, 2013, 787-838.
  4. Stasiuk, G. J.; Long, N. J. The Ubiquitous DOTA and Its Derivatives: The Impact of 1,4,7,10-Tetraazacyclododecane-1,4,7,10-Tetraacetic Acid on Biomedical Imaging. Chem. Commun. 2013, 49(27), 2732-2746.
  5. De León-Rodríguez, L. M.; Kovacs, Z. The Synthesis and Chelation Chemistry of DOTA−Peptide Conjugates. Bioconjugate Chem. 2008, 19(2), 391-402.
  6. Brechbiel, M. W. Bifunctional chelates for metal nuclides. The Quarterly Journal of Nuclear Medicine and Molecular Imaging 2008, 52(2), 166-173. (accessed Feb 7, 2019).
  7. Rogers, B. E.; Della Manna, D.; Safavy, A. In Vitro and In Vivo Evaluation of a 64Cu-Labeled Polyethylene Glycol-Bombesin Conjugate. Cancer Biotherapy and Radiopharmaceuticals 2004, 19(1), 25-34.
  8. Shi, J.; Kim, Y.-S.; Zhai, S.; Liu, Z.; Chen, X.; Liu, S. Improving Tumor Uptake and Pharmacokinetics of 64Cu-Labeled Cyclic RGD Peptide Dimers with Gly3 and PEG4 Linkers. Bioconjugate Chem. 2009, 20(4), 750-759.
  9. Li, L.; Turatti, F.; Crow, D.; Bading, J. R.; Anderson, A.-L.; Poku, E.; Yazaki, P. J.; Williams, L. E.; Tamvakis, D.; Sanders, P.; et al. Monodispersed DOTA-PEG-Conjugated Anti-TAG-72 Diabody Has Low Kidney Uptake and High Tumor-to-Blood Ratios Resulting in Improved 64Cu PET. Journal of Nuclear Medicine 2010, 51(7), 1139-1146.
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