T h e n e w e ng l a n d j o u r na l o f m e dic i n e

Cl inic a l I m pl ic a t ions of B a sic R e se a rch
Elizabeth G. Phimister, Ph.D., Editor

Click Here for Better Chemistry
Brian M. Zeglis, Ph.D., and Jason S. Lewis, Ph.D.
The drive to better understand living systems
and the pursuit of more effective clinical tools
have fueled a surge in research at the intersection of chemistry, biology, and medicine. A common motif in this work has been the modification of biologically active molecules to create
probes for the interrogation of biologic systems
and to produce diagnostics and therapeutics forthe clinic. Yet the chemical manipulation of biomolecules — whether small molecules, nucleic
acids, carbohydrates, lipids, proteins, or antibodies — is complicated by three challenges.
First, nature uses only a handful of functionalgroups (e.g., amines and carboxylic acids), eachwith its own demands when it comes to reactivity.
Second, a biomolecule can often contain multiple copies of a given functional group, and to
make matters worse, some of these can be situated in the area responsible for its biologic activity. As a result, modifying a biomolecule at asingle site without perturbing its function canbe a daunting task. Third, many biomolecules
are highly sensitive to temperature, solvent, andpH and can therefore be manipulated only underbiocompatible conditions. Historically, these intrinsic obstacles have combined to make thesynthesis of effective biomolecular tools arduousat best and impossible at worst.
The 2022 Nobel Prize in Chemistry wasawarded to Drs. Carolyn Bertozzi, Morten Meldal, and K. Barry Sharpless for their creation ofa suite of new chemical transformations —“click chemistry” — that has revolutionized research at the interface of chemistry and biology.
Click chemical reactions are fast, efficient, modular, clean, selective, and compatible with aqueous conditions. The name, coined by Sharpless,calls to mind molecular puzzle pieces that connect to one another (and only one another) simply and easily. Meldal and Sharpless independently

and concomitantly developed what is known
today as the canonical click reaction. In thistransformation, an azide and alkyne undergo acycloaddition reaction with the help of a copper
catalyst to form a triazole ring, thereby linking
together any cargoes attached to the members of
this molecular duo (Fig. 1A).1,2 One can easily
imagine how this copper-catalyzed azide–alkyne
cycloaddition (CuAAC) could be used to circumvent the aforementioned challenges: a biomolecule containing a single azide could easily be“clicked” to an alkyne-bearing partner under
mild, aqueous conditions without the formationof unwanted byproducts. Beyond chemical biology, the CuAAC ligation has proved indispensable in fields ranging from drug development
and materials chemistry to biomedical engineering and nanoscience.
Upon its discovery, the CuAAC reaction was
an instant hit, but some conditions proved unsuitable for the ligation. In complex biologicsystems, such as living organisms, it is just too
difficult to get the azide, alkyne, and catalyst
together in one place. A simpler, two-component
version of the reaction that was developed by
Sharpless and Meldal was needed. This is where
Bertozzi’s work comes in. Her variation on thecycloaddition still uses an azide but pairs it witha cyclic alkyne that is spring-loaded with whatchemists call “ring strain” (Fig. 1B).3 The releaseof this ring strain when the alkyne reacts withthe azide gives the cycloaddition the push itneeds to proceed without a copper catalyst. Thistransformation was dubbed the strain-promoted
azide–alkyne cycloaddition (SPAAC). As a bioorthogonal click reaction — a transformation thatcan occur in biologic systems without perturbing them — the SPAAC reaction has enabled theuse of click chemistry in previously inaccessible
biologic environments. This was elegantly demon-n engl j med﻿﻿  nejm.org﻿

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T h e n e w e ng l a n d j o u r na l o f m e dic i n e

A Copper-Catalyzed Click Reaction: CuAAC
Azide

N3

Cargo
1

Triazole

AlkyneCopper catalyst+Cargo
2

Cargo
1

NN NCargo
2

B Strain-Promoted Click Reaction: SPAAC
Azide

N3Cargo1F

FFF(No catalyst needed)+Cargo1Cargo2Strainedalkyne

Cargo
2N
N

N

Triazole

C CuAAC-Synthesized 18F-FET-βAG-TOCA
Radionuclide18F

OH

Triazole
N
H
N

N
N
O

H
N
O

O

Somatostatin
receptor–targeting
moiety

O

H
N

N
H

HN

S
H
N
O
OH

O

O

S

OH
O

H
N

N
H

O

Livermetastases

O

NH

O

NH2

NH

N
H

Bone metastases

OH

The radiolabeled imaging agent can be used to identify
certain neuroendocrine tumors, such as in this PET-CT
image of metastatic small-bowel neuroendocrine cancer.

D SPAAC-Synthesized Radiolabeled Monoclonal Antibody (89Zr-ssDFO-Pertuzumab) to Identify HER2-Positive Malignant Lesions

PertuzumabFab region
targets HER2
subdomain IILightchain

Heavy
chain

Azide

Glycan

Heavy
chain

Radionuclide

N3

Strainedalkyne+DFO(a radiometalchelator)

Thoracic nodelesionsLiverlesions

Triazole
N N
N

Radionuclide
89Zr
89Zr

An immunoPET image of metastasesin the liver and thoracic nodes.

2n engl j med

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Clinical Implications of Basic ResearchFigure 1 (facing page). When Chemistry Clicks.
Panel A shows a schematic diagram of the copper-­catalyzed azide–alkyne cycloaddition (CuAAC) reaction.
Panel B shows a schematic diagram of the strain-promoted azide–alkyne cycloaddition (SPAAC) reaction.
Panel C shows the structure of fluorine-18–labeled fluoroethyltriazole-Tyr3 –octreotate (18F-FET-βAG-TOCA)
and a transverse positron-emission tomographic–computed tomography (PET-CT) image (courtesy of Dr.
E.O. Aboagye, Imperial College London) acquired with18
F-FET-βAG-TOCA in a patient with metastatic smallbowel neuroendocrine cancer showing multiple liver
metastases. Panel D shows the use of the SPAAC reaction to modify azide-bearing sugars incorporated into
the heavy-chain glycans of pertuzumab with a chelator(desferrioxamine [DFO]) for subsequent radiolabelingwith zirconium-89 as well as a coronal PET image (courtesy of Dr. Randy Yeh, Memorial Sloan Kettering CancerCenter) acquired 6 days after the administration of theradioimmunoconjugate to a patient with human epidermal growth factor receptor 2 (HER2)–positive breast
cancer with multiple metastatic lesions in the liver andthoracic nodes. Fab denotes fragment antigen-binding.

strated by Bertozzi and colleagues when theytreated live zebrafish embryos with azide-modified sugars.4 As the embryos developed, theseazide-bearing sugars were metabolically incorporated into the cell-surface glycans of various
tissues, where they could then undergo SPAAC
reactions with strained alkynes labeled withfluorophores to illuminate the emerging glycome of the zebrafish.
The advent of click chemistry has had a seismic effect on almost all areas of chemical science. Our field — radiopharmaceutical chemistry — is no exception. Click chemistry is aremarkably powerful tool for radiochemistry:
the decay of radionuclides makes synthetic speed
a priority, and both selectivity and water compatibility are critical because biomolecules areoften used as platforms for radiotracers andradiotherapeutics. Over the past 15 years, theCuAAC and SPAAC reactions have been used tocreate radiopharmaceuticals labeled with everything from short-lived nuclides (e.g., carbon-11and fluorine-18 [18F]) for positron-emission tomography (PET) to long-lived isotopes (e.g., lutetium-177 and actinium-225) for targeted radionuclide therapy. Some of these agents have begun

to appear in the clinic. In 2016, 18F-labeled fluoroethyltriazole-Tyr3–octreotate, a somatostatin
receptor-targeting imaging agent that is synthesized with the CuAAC reaction, was translated
for the PET imaging of neuroendocrine tumors
in patients (Fig. 1C).5 More recently, we haveused the SPAAC reaction to create a variant of
pertuzumab that is radiolabeled with zirconium-89 on the heavy-chain glycans (Fig. 1D). A
trial of this probe for the antibody-based PET(immunoPET) imaging of human epidermal
growth factor receptor 2–positive malignant lesions in patients is currently under way (ClinicalTrials.gov number, NCT04692831).
This Nobel Prize marks an exciting and consequential moment for click chemistry. In thelaboratory, the trio of laureates have inspired thedevelopment of a host of new bioorthogonal
click reactions, each with its own applications inchemistry, biology, and medicine. Importantly,
click chemistry–based tools have increasingly
entered the clinic. The road from chemical discovery to medical application may be long, but
the technology that arose from the discoveries ofBertozzi, Meldal, and Sharpless is clearly well on
its way.
Disclosure forms provided by the authors are available withthe full text of this article at NEJM.org.
From the Department of Chemistry, Hunter College, City University of New York (B.M.Z.), the Department of Radiology(B.M.Z., J.S.L.) and the Program in Molecular Pharmacology(J.S.L.), Memorial Sloan Kettering Cancer Center, and the Department of Radiology, Weill Cornell Medicine (B.M.Z., J.S.L.)— all in New York.
This article was published on November 30, 2022, at NEJM.org.
1. Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse
chemical function from a few good reactions. Angew Chem Int
Ed Engl 2001;​40:​2004-21.
2. Meldal M, Tornøe CW. Cu-catalyzed azide-alkyne cycloaddition. Chem Rev 2008;​108:​2952-3015.
3. Agard NJ, Prescher JA, Bertozzi CR. A strain-promoted [3 + 2]
azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J Am Chem Soc 2004;​126:​15046-7.
4. Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR. In vivo
imaging of membrane-associated glycans in developing zebra­
fish. Science 2008;​320:​664-7.
5. Dubash SR, Keat N, Mapelli P, et al. Clinical translation of aclick-labeled 18F-octreotate radioligand for imaging neuroendocrine tumors. J Nucl Med 2016;​57:​1207-13.
DOI: 10.1056/NEJMcibr2213596
Copyright © 2022 Massachusetts Medical Society.

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