A new method to synthesize creatine derivatives

Article abstract of DOI:10.1007/s00726-013-1525-x

Creatine is an amino acid that has a pivotal role in energy metabolism of cells. Creatine acts as an ATP shuttle , carrying ATP to the sites where it is utilized, through its reversible phosphorylation by creatine kinase. Moreover, the creatine-phosphocreatine system delays ATP depletion during anoxia or ischemia, thus exerting a neuroprotective role during those pathological conditions. Thus, its administration has been advocated as a treatment or prevention of several conditions involving the central nervous system. However, creatine crosses poorly the blood-brain barrier and the cell plasma membrane, thus its administration has but a limited effect. The use of more lipophilic creatine derivatives has thus been suggested. However, such a synthesis is complicated by the intrinsic characteristics of the creatine molecule that hardly reacts with other molecules and easily cyclizes to creatinine. We obtained amide derivatives from creatine starting from a new protected creatine molecule synthesized by us, the so-called (Boc) ₂-creatine. We used a temporary protection only on the creatine guanidine group while allowing a good reactivity on the carboxylic group. This temporary protection ensured efficient creatine dissolution in organic solvents and offered simultaneous protection of creatine toward intramolecular cyclization to creatinine. In this manner, it was possible to selectively conjugate molecules on the carboxylic group. The creatine guanidine group was easily released from the protection at the end of the reaction, thus obtaining the desired creatine derivative.

Full text of DOI:10.1007/s00726-013-1525-x

Amino Acids (2013) 45:821–833
DOI 10.1007/s00726-013-1525-x
ORIGINAL ARTICLE
A new method to synthesize creatine derivatives
•
Patrizia Garbati Annalisa Salis Enrico Adriano
•
•
•
Andrea Galatini Gianluca Damonte
•
•
Maurizio Balestrino Enrico Millo
Received: 4 April 2013 / Accepted: 23 May 2013 / Published online: 7 June 2013
Ó Springer-Verlag Wien 2013
Abstract Creatine is an amino acid that has a pivotal role
in energy metabolism of cells. Creatine acts as an ‘‘ATP
shuttle’’, carrying ATP to the sites where it is utilized,
through its reversible phosphorylation by creatine kinase.
Moreover, the creatine-phosphocreatine system delays
ATP depletion during anoxia or ischemia, thus exerting a
neuroprotective role during those pathological conditions.
Thus, its administration has been advocated as a treatment
or prevention of several conditions involving the central
nervous system. However, creatine crosses poorly the
blood–brain barrier and the cell plasma membrane, thus its
administration has but a limited effect. The use of more
lipophilic creatine derivatives has thus been suggested.
However, such a synthesis is complicated by the intrinsic
characteristics of the creatine molecule that hardly reacts
with other molecules and easily cyclizes to creatinine. We
obtained amide derivatives from creatine starting from a
new protected creatine molecule synthesized by us, the so-
called (Boc)2-creatine. We used a temporary protection
only on the creatine guanidine group while allowing a good
reactivity on the carboxylic group. This temporary pro-
tection ensured efficient creatine dissolution in organic
solvents and offered simultaneous protection of creatine
toward intramolecular cyclization to creatinine. In this
manner, it was possible to selectively conjugate molecules
on the carboxylic group. The creatine guanidine group was
easily released from the protection at the end of the reac-
tion, thus obtaining the desired creatine derivative.
Keywords Creatine ꢀ (Boc)2-creatine ꢀ Creatine
derivatives synthesis ꢀ Guanidine group ꢀ Creatine
reactivity
P. Garbati ꢀ E. Adriano ꢀ M. Balestrino
Department of Neuroscience, Ophthalmology, Genetics,
Maternal–Infantile Sciences, University of Genova,
Largo Paolo Daneo 3, 16132 Genoa, Italy
Introduction
A. Salis
Creatine is an amino acid that has a pivotal role in energy
metabolism of cells. It is reversibly phosphorylated to
phosphocreatine (Andres et al. 2008), creating a creatine/
phosphocreatine system that acts as an ‘‘adenosine tri-
phosphate (ATP) shuttle’’, carrying ATP to the sites where
it is utilized (Greenhaff 2001), an action so important that
hereditary conditions where brain creatine is absent or
reduced cause severe brain malfunction (Nasrallah et al.
2010; Schulze 2003). Moreover, the creatine phosphocre-
atine system delays ATP depletion during anoxia or
ischemia, thus exerting a neuroprotective role during those
pathological conditions (Balestrino et al. 2002). Thus, its
administration has been advocated as a treatment or pre-
vention of several conditions involving the central nervous
Department of Hearth Environmental and Life Science
(DISTAV), University of Genova, Corso Europa 26,
16132 Genoa, Italy
A. Salis ꢀ G. Damonte ꢀ E. Millo
Center of Excellence for Biomedical Research, University
of Genova, Viale Benedetto XV 5, 16132 Genoa, Italy
A. Galatini
Department of Chemistry and Industrial Chemistry, University
of Genova, Via Dodecaneso 31, 16146 Genoa, Italy
G. Damonte ꢀ E. Millo (&)
Department of Experimental Medicine, Section of Biochemistry,
University of Genova, Viale Benedetto XV 1, 16132 Genoa,
Italy
e-mail: enrico.millo@unige.it
123

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P. Garbati et al.
system, the main ones being creatine deficiency syndromes
(Nasrallah et al. 2010; Schulze 2003), anoxia or ischemia
(Perasso et al. 2013), neurodegenerative diseases (Adhih-
etty and Beal 2008; Beal 2011). However, creatine crosses
poorly the blood–brain barrier (Perasso et al. 2003) and the
cell plasma membrane (Lunardi et al. 2006) despite the
presence of a specific transporter (Ohtsuki et al. 2002;
Snow and Murphy 2001), thus its administration has but a
limited effect. The use of more lipophilic creatine deriva-
tives has thus been suggested (Lunardi et al. 2006; Perasso
et al. 2008; Perasso et al. 2013).
overcome by its conversion to an appropriate salt form.
Infact the addition of particular lipophilic counterion rep-
resents a simple and convenient procedure for dissolution
of creatine and other different guanidino or amidino com-
pounds in organic solvents. In our experience, this method
gives good results only with substances at high nucleo-
philicity (example with some aliphatic or cyclic amine),
moreover this strategy involves low yields and long times
of purification.
Moreover, the synthesis of creatine derivatives can be
performed starting from a secondary amine called sarco-
sine. These conjugates can be synthesized by the guanid-
inylation of sarcosyl derivatives. This strategy comprises
different stages, but the yields of the products in literature
are quite low (Burov et al. 2011a).
To this aim, we decided to modify the creatine molecule
to allow it to cross the phospholipid bilayer of biological
membranes without the use of its specific transporter.
However, modifying the creatine molecule is difficult
because it hardly reacts with other molecules because of its
intrinsic characteristics. Creatine is a guanidine zwitter-
ionic molecule that, forming a sort of internal salt, binds
with difficulty with other compounds.
To overcome these problems, and to ameliorate the
creatine reactivity, our strategy was to design a creatine
molecule with a temporary protection only on the guani-
dine group (Powell et al. 2003; Robinson and Roskamp
1997) while allowing a good reactivity on the carboxylic
group. Indeed, this temporary protection ensured efficient
creatine dissolution in organic solvents offering simulta-
neous protection of creatine toward intramolecular cycli-
zation to creatinine. In this manner, it was possible to
conjugate selectively several molecules with different
characteristics, on the carboxylic group while the guanidine
group had to be easily released from the protecting group to
allow the final molecule to be phosphorylated upon
reaching the site of action.
Creatine solubility is a key aspect relative to its ability to
react with other compounds. Under normal conditions,
creatine monohydrate exhibits low aqueous solubility
(Singh and Dash 2009; Windholz et al. 1976). The aqueous
solubility increases with the increase of temperature; even
at 60 °C a great abundance of sub-products is present
(Pischel and Gastner 2008).
At a pH between 3 and 12, creatine is principally in its
zwitterionic form (Edgar and Shiver 1925) and this fact
adversely affects the bonds with other molecules. An acidic
environment is the best condition to promote the creatine
solubility, such that at pH \ 3 creatine is mainly present as
a cation. Furthermore, the creatine shows low solubility in
a large part of organic solvents (Windholz et al. 1976).
Preparations in which the creatine is linked to other
compounds by forming a salt are well documented (Ne-
grisoli and Del Corona 1999; Pischel et al. 1999). It is
necessary that these compounds keep the undissociated
form during the passage through biological membranes so
that such compound can use the carrier of the molecule
linked to creatine or the characteristics that the lipophilic
molecule partner offers. In addition, there are other data on
the creatine prodrug where it is covalently linked in the
form of esters or anhydrides (Burov et al. 2011b; Heuer
et al. 2009; Peters et al. 2007) with different substances.
Although creatine esters are more lipophilic than crea-
tine molecule, they are not stable enough and their enzy-
matic or non-enzymatic cleavage results in the formation of
creatinine instead of creatine release (Adriano et al. 2011).
As an alternative the possibility of a covalent link between
creatine and other substances was investigated; this was
done by exploiting direct acylation of the compound with
particular creatine salts (Burov et al. 2011a). Using this
strategy, the problem of solubility of creatine can be
We therefore decided to build a creatine with the gua-
nidine group temporarily protected; tert-butoxycarbonyl
group (t-Boc) was the most suitable protective group found
for the purpose. The protection with t-Boc also provided a
high solubility of the molecule in organic solvents, more-
over, allowing a high reactivity of the molecule on the
carboxyl group.
Starting from this particular kind of creatine, called
(Boc)2-creatine, some creatine derivatives with different
aminogroups in high yield and purity were obtained.
Materials and methods
All chemicals were purchased from Sigma-Aldrich and
used as received without further purification. All untreated
solvents used were of HPLC grade.
The thin layer chromatography (TLC) was performed on
UV plates (Fluka Analytical, Silica on TLC Alu foils, with
fluorescent indicator 254 nm). Four different eluent phases
were used for the TLC: n-butanol:acetic acid:water (6:2:2),
MeOH:CHCl3:CH3COOH (8:1.75:0.25), CH2Cl2:MeOH:
CH3COOH (9.25:0.5:0.25) and hexane:ethyl acetate (5:5).
123

Method to synthesize creatine derivatives
823
Revelation: solution of ninhydrin (0.2 g) in ethanol (50 ml)
was 0.1 % formic acid (HCOOH) in water A and 0.1 %
HCOOH in acetonitrile (B).
spraying before putting on hot plate over 100 °C.
Electrospray ionization mass spectrometry and high
performance liquid chromatography coupled
to electrospray mass spectrometry methods
Nuclear magnetic resonance (NMR) analysis method
1H-NMR and 13C-NMR spectra were recorded on a Varian
MercuryPlus spectrometer at 300 and 75 MHz, respec-
tively. Chemical shifts are reported as d values (ppm).
Synthesis of N1,N2-bis(tertbutoxycarbonyl)-N3-methyl-
guanidino-N3-acetic acid ((Boc)2-creatine).
The analysis of the crude products was performed by
electrospray ionization mass spectrometry (ESI-MS) full
scan and MS2 analysis (ESI-MS2) were carried out in
positive ion mode on an Agilent 1100 series LC/MSD ion
trap XCT instrument (Agilent Technologies, Palo Alto,
CA, USA) in direct infusion (DIA) using the infusion pump
NE1000 (KF Technology, Roma, Italy). The flow rate was
set at 5 ll/min. Each product was diluted in acetonitrile:
water 50:50 containing 0.1 % formic acid to get a final
concentration of 0.01 mg/ml.
Method A using N1,N2-Bis(tert-butoxycarbonyl)-S-
methylisothiourea as guanylating agent
To a solution of sarcosine ethyl ester (1 g; 6.5 mmol;
1.2 equivalents), N1,N2-Bis(tert-butoxycarbonyl)-S-meth-
ylisothiourea (1.575 g; 5.4 mmol; 1 equivalent), and tri-
ethylamine (16.3 mmol; 2.27 ll; 3 equivalents) in 10 ml
anhydrous dimethylformamide (DMF) was added HgCl2
(1,620 g; 6 mmol; 1.1 equivalents). The suspension was
stirred at room temperature for about 18 h. Then the
crude reaction mixture was taken up in diethyl ether, a
white precipitate was formed and then filtered through a
filter vacuum. The filtrate was washed two times with
deionized water and after other three times with a
solution of NaCl 0.1 M. The organic phase was evapo-
rated to dryness and finally lyophilized. Analysis of the
crude product was performed by ESI-MS. The result
showed the expected molecular weight of N1,N2-
bis(tertbutoxycarbonyl)-N3-methylguanidino-N3-acetic
acid ethyl ester.
The qualitative analysis of creatine derivatives and the
(Boc)2-creatine stability were carried out on an Agilent
1100 HPLC system coupled to a MSD Ion Trap XCT mass
spectrometer, equipped with an electrospray ion source
(HPLC–ESI-MS). Separations were performed on a HILIC
silica 2.1 9 150 mm2 Atlantis column with 3-lm particle
size (Waters Corporation, Milford, MA, USA). Eluents
were acetonitrile (A) and 50 mM ammonium acetate
(B) whose pH was adjusted to 5 with acetic acid. The flow
rate was set to 0.20 ml/min. The column temperature was
set at 30 °C. The best chromatographic conditions were
outlined as follows: injection at 80 % A, hold for 3 min,
then decrease by step to 50 % A at 3.01 min and hold for
1 min, then reconditioning to initial conditions at 4.01 min
and hold for 12 min. Injection volume was 5 ll. Ions were
detected in ion-charged control with a target ions value of
50,000 and an accumulation time of 300 ms, using the
following operation parameters: capillary voltage:
3,000 V; nebulizer pressure: 40 psi; drying gas: 10 L/min;
dry temperature: 325 °C; rolling averages 3, averages 8.
Mass spectra were acquired in positive ion mode in the
100–400 m/z mass range. An amplitude voltage of 1.0 V
was typically used for fragmentation in the ion trap MS2
analysis.
A solution of acetonitrile and NaOH 1 N 1:1 was added
and the mixture was stirred at room temperature. The
reaction was carried out until the complete hydrolysis of
the ethyl group, monitored by TLC, to obtain the desired
compound. At the end the pH was adjusted to 6 using HCl
1 N. The compound obtained was dried in vacuum to
obtain a white powder and then purified by preparative RP-
HPLC. Fractions containing the molecule were collected,
vacuum dried and finally lyophilized to afford colorless
powder of N1,N2-bis(tertbutoxycarbonyl)-N3-methylguani-
dino-N3-acetic acid. Yield 86 %. The compound purity was
finally verified by HPLC–MS analysis as described above.
The qualitative analysis of the product was finally con-
firmed by ESI-MS and ESI-MS2 analysis.
1H NMR (300 MHz, CDCl3) d 9.54 (s, broad 1H), 3.89
(s, 2H), 3.01 (s, 3H), 1.44 (s, 18H).
13C NMR (75 MHz, CDCl3) d 175.43, 161.53, 154.63,
151.53, 81.72, 79.66, 55.05, 38.41, 28.34.
ESI-MS: m/z 332.00 [M ? H]?; ESI-MS2: m/z (rel.
int.): 275 [M-C4H9]? (100); 220 [M-2 9 C4H9]? (40);
176 [M-(2 9 C4H9)-CO2] ? (10).
Preparative HPLC method
The products were purified by preparative reverse phase
high performance liquid chromatography (RP-HPLC) on a
Shimadzu LC-9A preparative HPLC equipped with a
Phenomenex C18 Luna column 21.20 id 9 250 mm
(Phenomenex Italia, Castel Maggiore, Bologna, Italy).The
separation was performed in gradient starting with 10 %
solvent B for 5 min, linearly increasing to 70 % solvent B
in 30 min and up to 100 % B in 10 min. The solvent used
123

824
P. Garbati et al.
CH3
NH
S
O
O
Method A
Method B
N
HgCl2
O
O
O
H
O
O
N
N
+
O
O
N
O
N
O
NH
N
O
O
O
N
NH
O
O
CH3CN:NaOH
(1:1)
O
O
O
N
OH
O
NH
N
O
Fig. 1 (Boc)2-Creatine synthesis. Sarcosine ethyl ester reacts
with N1,N2-Bis(tert-butoxycarbonyl)-S-methylisothiourea
creatine ethyl ester that is subjected to basic hydrolysis leading to the
formation of (Boc)2-creatine, able to react with other molecules
through its carboxyl group
(method
A) or with N1,N2-Bis(tert-butoxycarbonyl)-1H-pyrazole-1-carboxa-
midine (method B) as guanylating agent. This reaction gives (Boc)2-
Method B using N1,N2-Bis(tert-butoxycarbonyl)-1H-
The qualitative analysis of the product was finally
confirmed by ESI-MS and ESI-MS2 analysis.
pyrazole-1-carboxamidine as guanylating agent
1H NMR (300 MHz, CDCl3) d 9.54 (s, broad 1H), 3.89
(s, 2H), 3.01 (s, 3H), 1.44 (s, 18H).
To a solution of sarcosine ethyl ester (1 g; 6.5 mmol; 1.2
equivalents), in 10 ml of anhydrous DMF, N1,N2-Bis(tert-
13C NMR (75 MHz, CDCl3) d 175.43, 161.53, 154.63,
151.53, 81.72, 79.66, 55.05, 38.41, 28.34.
butoxycarbonyl)-1H-pyrazole-1-carboxamidine
(1.68 g;
ESI-MS: m/z 332.00 [M ? H]?; ESI-MS2: m/z (rel.
int.): 275 [M-C4H9]? (100); 220 [M-C8H18]? (40).
ESI-MS: m/z 332.00 [M ? H]?; ESI-MS2: m/z (rel.
int.): 275 [M-C4H9]? (100); 220 [M-2 9 C4H9]? (40);
176 [M-(2 9 C4H9)-CO2] ? (10).
5.4 mmol; 1 equivalent), and triethylamine (2.26 ml;
16.25 mmol; 3 equivalents) were added. The suspension
was stirred at room temperature for about 48 h. Then the
crude reaction mixture was centrifuged at 4,800 rpm for
10 min. The supernatant obtained was taken up in diethyl
ether and washed three times with deionized water. The
organic solution was evaporated to minimum volume and
finally lyophilized. Analysis by ESI-MS was performed.
The result showed the expected molecular weight of N1,N2-
bis(tertbutoxycarbonyl)-N3-methylguanidino-N3acetic acid
ethyl ester.
Synthesis of amino acids esters
Some of amino acids esters were purchased; others have
been esterified by us following the methods in literature
with some modifications (Chen et al. 1999; Li and Sha
2008).
A solution of acetonitrile and NaOH 1 N 1:1 was added
and the mixture was stirred at room temperature. The
reaction was carried out until the complete hydrolysis of
the ethyl group, monitored by TLC, to obtain the desired
compound. At the end the pH was adjusted to 6 using HCl
1 N. The compound was evaporated to dryness under
vacuum and then purified by preparative RP-HPLC. Frac-
tions containing the molecule were collected, vacuum dried
and finally lyophilized to afford colorless powder of N1,N2-
bis(tertbutoxycarbonyl)-N3-methylguanidino-N3-acetic acid.
Yield 83 %. The compound purity was finally verified by
HPLC–MS as described above.
Briefly, the amino acid (1 equivalent) was suspended in
chlorotrimethylsilane (2 equivalents) and a suitable volume
of methanol was added. The reaction mixture was stirred at
room temperature monitoring the reaction with TLC
analysis (MeOH:CHCl3:CH3COOH 8:1.75:0.25). The
reaction was carried out for 12–48 h, depending on the
amino acid, and mainly monitored until the reaction was
complete. The organic solution was then evaporated under
vacuum and finally repeatedly lyophilized to obtain the
final product. The qualitative analysis of the products was
123

Method to synthesize creatine derivatives
825
Creatine–valine ethyl ester
confirmed by ESI-MS analysis. The aminoacid esters were
used without further purification.
The product was obtained as a white powder in 76 % yield.
Rf = 0.34 (MeOH:CHCl3:CH3COOH 8:1.75:0.25). ESI-
MS: m/z 259.08 [M ? H]?; ESI-MS2: m/z (rel. int.): 241
[M-H2O]? (5); 114 [M-C7H15NO2]? (100).
General procedure for synthesis of creatine–amino
acids esters
1H NMR (300 MHz, H2O/D2O 9/1) d 8.40 (d, broad
J = 6.9 Hz, 1H), 6.75 (s, broad 3H), 4.20–3.98 (m, 5H),
2.86 (s, 3H), 2.14–1.94 (m, 1H), 1.10 (t, J = 7.1 Hz, 3H),
0.78 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H).
13C NMR (75 MHz, H2O/D2O 9/1) d 173.39, 169.54,
157.87, 62.51, 58.86, 52.30, 36.97, 29.79, 18.12, 17.15,
13.24.
(Boc)2-creatine (1 equivalent), was dissolved in anhydrous
DMF. Isobutyl chloroformate (1 equivalent) and N-meth-
ylmorfoline (1 equivalent) were added to this compound
while holding the reaction at 0 °C. After 10 min the
reaction was held at room temperature protected from light.
At this point, amino acid ester (1.5 equivalents) was pre-
pared by stirring commercially available or synthesized
ester hydrochloride and triethylamine (1.5 equivalents) in
dry DMF for 30 min. The mixture was centrifuged to
remove the resulting salt, before adding it to the activated
(Boc)2-creatine. The solution was stirred for 24–48 h
depending on the aminoacid used monitoring the reaction
by TLC. When the reaction was completed the mixture was
centrifuged and the supernatant was lyophilized. After that,
the compound was dissolved in diethyl ether or ethyl
acetate depending on the aminoacid used, and the organic
solution was washed three times with deionized water.
Indeed the organic phase was evaporated under vacuum
until dryness. The crude product was then purified by
preparative RP-HPLC.
Creatine–threonine methyl ester
The product was obtained as a white powder in 85 % yield.
Rf = 0.39 (n-butanol:acetic acid:water 6:2:2). ESI-MS:
m/z 247.08 [M ? H] ? ; ESI-MS2: m/z (rel. int.): 229
[M-H2O] ? (5); 114 [M-C5H11NO3] ? (100).
1H NMR (300 MHz, H2O/D2O 9/1) d 8.39 (d, broad
J = 8.3 Hz, 1H), 6.77 (s, broad 3H), 4.42 (dd, J = 8.3,
2.8 Hz, 1H), 4.25 (dd, J = 6.3, 2.8 Hz, 1H), 4.12 (s, 2H),
3.62 (s, 3H), 2.89 (s, 3H), 1.03 (d, J = 6.3 Hz, 3H).
13C NMR (75 MHz, H2O/D2O 9/1) d 172.21, 169.83,
157.87, 66.94, 58.31, 53.00, 52.43, 37.01, 18.68.
A solution of dichloromethane (DCM) and trifluoro-
acetic acid (TFA) 1:1 was indeed added to the purified
product at 0 °C. When the reaction was completed the
compound was treated with ice cold diethyl ether. If a
precipitate was formed, it was separated by centrifugation
(4,800 rpm per 10 min), washed with diethyl ether and
finally lyophilized to obtain a solid or oil. In absence of any
precipitate the organic mixture was washed twice with
deionized water and then evaporated under vacuum. The
final derivative was then lyophilized to obtain a powder or
oil. The compounds purity was finally verified by HPLC–
MS analysis as described above. The qualitative analysis of
the products was finally confirmed by ESI-MS and ESI-
MS2 analysis.
General procedure for synthesis of creatine–amides
(Boc)2-creatine (1 equivalent) was dissolved in anhydrous
DMF. Isobutyl chloroformate (1 equivalent) and N-meth-
ylmorfoline (1 equivalent) were added to this compound
holding the reaction at 0 °C. After 10 min the reaction was
held at room temperature protected from light. At this
point, a solution of amine (1.5 equivalents) in anhydrous
DMF was added to the activated (Boc)2-creatine. The
mixture was stirred for 24–48 h, depending on the amine,
monitoring the reaction by TLC. When the reaction was
completed, the mixture was centrifuged and the supernatant
was lyophilized. The compound was dissolved in diethyl
ether and then washed three times with deionized water.
Indeed the organic phase was evaporated under vacuum
until dryness. The crude product was then purified by
preparative RP-HPLC.
Creatine–alanine methyl ester
The product was obtained as a white powder in 84 % yield.
Rf = 0.41 (MeOH:CHCl3:CH3COOH 8:1.75:0.25). ESI-
MS: m/z 217.08 [M ? H]?; ESI-MS2: m/z (rel. int.): 200
[M-H2O]? (5); 114 [M-C4H9NO2]? (100).
1H NMR (300 MHz, H2O/D2O 9/1) d 8.49 (d, broad
J = 5.1 Hz, 1H), 6.76 (s, broad 3H), 4.39–4.22 (m, 1H),
4.03 (s, 2H), 3.59 (s, 3H), 2.87 (s, 3H), 1.26 (d, J = 7.3 Hz,
3H).
13C NMR (75 MHz, H2O/D2O 9/1) d 174.82, 169.52,
157.79, 52.82, 52.21, 48.63, 36.87, 15.79.
A solution of DCM and TFA 1:1 was indeed added to
the purified product at 0 °C. When the reaction was com-
pleted the compound was treated with ice cold diethyl
ether. If a precipitate was formed, it was separated by
centrifugation (4,800 rpm per 10 min), washed with die-
thyl ether and finally lyophilized to obtain a solid or an oil.
In absence of any precipitate, the organic mixture was
washed twice with deionized water and then evaporated
under vacuum. The final derivative was then lyophilized to
123

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P. Garbati et al.
obtain a powder or an oil. The qualitative analysis of the
product was finally confirmed by ESI-MS analysis.
The reason for this poor reactivity is probably due to the
hydrogen bonds that are formed between the carboxyl
group and the two amino groups of the guanidine moiety.
In a molecule in which the guanidine group is protected,
you can activate the carboxyl group with the same acti-
vating reagents.
Stability of (Boc)2-creatine in aqueous solution
For the measurement of the (Boc)2-creatine stability the
molecule was dissolved, at a final concentration of 2 mM,
in artificial cerebrospinal fluid (ACSF) at 36 °C and the
solution was diluted 400 times in eluent A:B (80:20 %).
The ACSF had the following composition: NaCl 130 mM,
KCl 3.5 mM, NaH2PO4 1.25 mM, NaHCO3 24 mM, CaCl2
2.4 mM, MgSO4 1.2 mM, glucose 10 mM. Then, 5 ll of
solution was injected in HPLC–MS system, and the dif-
ferent peaks were quantified. We were interested to know
the amount of (Boc)2-creatine that was still intact at time 0
and after 5, 60, 120,150 and 180 min of treatment. Thus, at
these times, the area under each peak was measured, and
normalized as a percentage of the area of (Boc)2-creatine
peak at time 0 min.
Initially, we tried using some temporary protective
groups for the guanidine group described in the literature:
2,2,4,6,7,pentamethyldihydrobenzofuran-5-sulfonyl group
(Pbf), 4-toluensulfonyl group (Tosyl) tryphenylmethyl
group (Trt) and others (Carpino et al. 1993; Isidro-Llobet
et al. 2009; Ruskin 1946), but these were not convenient
because attempts to clamp the guanidine group lead to
constructs with too low yields. Indeed, even using the Pbf
blocking group the product was completely degraded when
put in an acid environment intended to remove the pro-
tective group. The best protecting group found was t-Boc
which binds to both the nitrogen atoms of the guanidine
group. Heuer et al. (2009) describe a method to synthesize
(Boc)2-creatine starting from creatine itself using agents
able to insert the t-Boc group such as tert-butoxycarbonyl
anhydride. Following this protocol, we did not find the
desired intermediate because of the instability and low
solubility of creatine at the pH of the experimental
procedure.
Results and discussion
The chemical synthesis of creatine derivatives is quite
difficult because of its intrinsic characteristics. Normally,
creatine hardly reacts with other molecules because of
zwitterionic form which creates a sort of internal salt,
decreasing its reactivity.
Therefore, we synthesized the (Boc)2-creatine, which is
a form of creatine where the two nitrogen atoms of its
guanidine group are protected by two t-Boc groups. This
form of creatine has several advantages: it is soluble in
organic solvents, reacts easily and it is stable and does not
degrade in creatinine. This particular derivative cannot be
synthesized starting from creatine itself, but using sarco-
sine a similar compound lacking the guanidine moiety. The
first step of the process involved the use of sarcosine ethyl
ester as a precursor which was converted to (Boc)2-creatine
by the use of a guanylating agent which was protected on
both its nitrogen atoms with t-Boc. This allowed its direct
synthesis. Two methods were used to synthesize the
(Boc)2-creatine using two different guanylating agents; the
two reactions were more or less similar. The sarcosine
ethyl ester was easily converted to a protected guanidine
compound through a simple procedure. Introducing a
guanidine group to sarcosine ethyl ester gives a higher
yield if the guanylating agent used already has the t-Boc
protection. The same reaction without t-Boc on the
guanylating agent gives a very low yield. The guanylating
agents used were N1,N2-Bis(tert-butoxycarbonyl)-S-meth-
ylisothiourea (Powell et al. 2003) and N1,N2-Bis(tert-
butoxycarbonyl)-1H-pyrazole-1-carboxamidine (Robinson
and Roskamp 1997). The yields obtained with these two
guanylating agents were essentially the same. The final
ethyl ester of (Boc)2-creatine was easily isolated in high
yield.
Our goal was to synthesize creatine derivatives able to
cross the phospholipid bilayer of biological membranes
without using the specific transporter of creatine. Creatine
has two possible groups able to form bonds with other
molecules: carboxylic and guanidine groups.
Creatine derivatives with drastic alteration of guanidine
nitrogen present a high degree of lipophilicity but their
function in the brain may have negative aspects such as the
impediment of the creatine phosphorylation. At this regard,
it is necessary that the molecule that we are going to
combine selectively is covalently bound with the creatine
carboxylic group while the guanidine group must be free to
interact with the creatine kinase enzyme.
Our strategy was to design a creatine molecule with a
temporary protection only on the guanidine group that
allows us to obtain a new precursor which had both a high
solubility in organic solvents and good reactivity of the
carboxylic part.
Moreover, the carboxyl group could not be activated
with standard activating agents like carbodiimide such as
dicyclohexylcarbodiimide (DCC) or ethyl-(N’,N’-dimeth-
ylamino)propylcarbodiimide hydrochloride (EDC) or
aminium-based reagents such as O-(benzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium
hexafluorophosphate
(HBTU) if the guanidine group was free.
123

Method to synthesize creatine derivatives
827
In the next step of the procedure, the ester of (Boc)2-
creatine was subjected to alkaline hydrolysis to form the
(Boc)2-creatine (Fig. 1). The method advantageously
allowed to obtain a good yield ([80 %) and excellent
purity of the final product as shown in the ESI-MS and
ESI-MS2 spectra of Fig. 2.
creatine, by conjugating a molecule having a functional
group capable of reacting with the free carboxyl group of
creatine. Due to their greater stability, we preferred to
synthesize amide derivatives of creatine. In the Table 1
was summarized all the derivatives of creatine synthesized.
We obtained various compounds where the creatine is
linked to amino acids or amines with yields ranging from
35 to 85 % depending on the type of aminoacid or amine
used. The compound containing histidine has a low yield
probably due to the poor reactivity of amino group of
histidine (see Table 1).
In addition, we tested the (Boc)2-creatine stability. We
found that this molecule has a great stability, indeed after
6 months at room temperature about 100 % of the product
was present. Its stability is also evaluated in an aqueous
medium, ACSF: after 3 h 96.6 % of the initial value was
still present. The stability was verified using ACSF because
this is the incubation medium that we will use in future
biological experiments. Furthermore the brain is the target
for these kinds of molecules where the aqueous medium
present is the cerebrospinal fluid. The (Boc)2-creatine was
subsequently used to synthesize various derivatives of
The reaction between the carboxylic group and the
amino group of amino acids or amines obviously requires
the activation of the carboxyl group. We tried different
standard activating agents as DCC, EDC and HBTU and
others. Even if these agents allow us to obtain the coupling
of amino groups the final yield of the entire process is low
Fig. 2 ESI-MS and ESI-MS2
spectra of (Boc)2-creatine.
a Protonated (Boc)2-creatine
molecular ion (m/z 332.00).
b (Boc)2-Creatine ESI-MS2
spectra (see details in
‘‘Materials and methods’’
section)
123

828
P. Garbati et al.
Table 1 Synthesis of creatine derivatives starting from (Boc)2-creatine
O
O
N
Isobutyl
O
O
N
chloroformate,
NH
DCM:TFA
(1:1)
N-methylmorpholine
O
N
R
R
O
N
R
N
HN
OH
+
H2N
N
O
NH
N
R1
R1
O
NH
O
R1
O
O
MW
Creatine derivatives structure
CH3
Yield
1
2
Creatine-Alanine
methyl ester
216.23
301.35
CH3
O
O
H2N
N
84%
CH3
NH
O
NH
Creatine-Arginine
methyl ester
H2N
HN
NH
65%
CH3
N
O
O
H2N
CH3
NH
O
CH3
NH
3
Creatine-Leucine
methyl ester
258.31
H3C
NH
CH3
N
O
O
68%
82%
O
H2N
H2N
CH3
CH3
O
NH
4
5
Creatine-Glycine
methyl ester
202.21
273.33
CH3
N
H
O
NH
O
NH
Creatne-Lysine
methyl ester
NH2
78%
CH3
N
O
O
H2N
CH3
NH
O
NH
On the top is described the synthetic pathway for the preparation of the various derivatives. The creatine derivatives synthesized are summarized
in the table with their molecular weight and yield
123

Method to synthesize creatine derivatives
829
6
Creatine-Tyrosine
methyl ester
308.33
OH
66%
CH3
N
O
O
O
O
H2N
H2N
CH3
CH3
NH
NH
O
O
NH
NH
7
8
Creatine-Serine
methyl ester
232.23
282.29
HO
CH3
N
74%
28%
Creatine-Histidine
methyl ester
N
NH
CH3
N
O
O
O
O
H2N
H2N
CH3
CH3
NH
O
NH2
NH
NH
9
Creatine-Asparagine
methyl ester
259.26
276.35
O
CH3
N
46%
66%
NH
O
S
10 Creatine-Methionine
methyl ester
H3C
CH3
N
O
O
H2N
CH3
NH
O
H3C
NH
11 Creatine-Threonine
methyl ester
246.26
CH3
O
OH
H2N
N
NH
85%
O
NH
O
CH3
123

830
P. Garbati et al.
12 Creatine-Proline
methyl ester
242.27
258.32
CH3
O
N
O
CH3
N
O
62%
H2N
NH
CH3
13 Creatine-Valine ethyl
ester
O
H3C
CH3
H2N
N
NH
O
76%
NH
O
H3C
14 Creatine-
Phenylalanine methyl
ester
292.34
CH3
CH3
N
O
O
O
H2N
NH
82%
NH
15 Creatine-γ-
Aminobutyric acid
methyl ester
230.26
220.27
198.26
186.25
CH3
O
O
H2N
N
68%
52%
59%
NH
O
CH3
O
NH
16 Creatine-p-Toluidine
17 Creatine-Piperidine
CH3
N
CH3
H2N
NH
CH3
NH
O
H2N
N
N
NH
18 Creatine-
Diethylamine
CH3
O
H2N
N
N
CH3
38%
NH
H3C
123

Method to synthesize creatine derivatives
831
19 Creatine-2-amino-4-
picoline
221.26
CH3
N
CH3
N
O
35%
H2N
N
H
NH
20 Creatine-3-
252.33
CH3
O
(methylthio)aniline
CH3
H2N
N
42%
S
N
H
NH
and the contemporary presence of sub-products discour-
ages their use.
kinase enzyme. The final construct had to be released from
the t-Boc protection to become active. But often, the t-Boc
removal in acidic environment could cause the degradation
of the molecule with the formation of sub-products, so this
step is crucial in the entire protocol.
The mixed anhydride method with isobutyl chlorofor-
mate was most advantageous in terms of reaction com-
pletion and simplicity of the subsequent purification. Better
results were obtained by using 1 equivalent of isobutyl
chloroformate and dropwise addition of 1 equivalent of
N-methylmorfoline to the reaction mixture of (Boc)2-cre-
atine followed by the addition of the amino component.
The entry sequence of the various reagents was funda-
mental. It prevented the formation of the side reaction that
gives a urethane by-product and liberates the amino acid or
the amine group. These products were formed if the amino
group was added before using the alkyl chloroformate
(Chaudhary et al. 2003).
We tried different methods to detach the t-Boc groups
from the guanidine group of creatine to select the optimal
condition. We used TFA/DCM (1:1) at 0 °C, H2O/TFA
(1:1) at room temperature, HCl 1 N at room temperature
and CH3COCl:MeOH (1:1) at 0 °C (Cheguillaume et al.
2001; Nudelman et al. 1998; Zhu et al. 2000). Only the first
method detached the two t-Boc groups on creatine pro-
viding the final product without further problems.
Deprotection by TFA/DCM at 0 °C seemed to be the
method of choice to obtain the desired product without
forming creatinine or other sub-products in all the deriva-
tives tested.
In our experience, we also observed that the addition of
the amino acid in its hydrochloride form gives a low yield
or in same cases even absence of the reaction. To overcome
this problem, we transformed the hydrochloride form into
an amino acid free form (Todoroki et al. 2011), thus
increasing the amount of the final product.
As an example, we characterized three specific creatine
derivatives (creatine–alanine methyl ester, creatine–valine
ethyl ester, creatine–threonine methyl ester). In addition to
HPLC–MS analysis, performed on the derivatives descri-
bed in table 1, for these three derivatives we also reported
NMR and ESI-MS2 analysis. We chose to deepen the
analysis on such compounds because we will use them in
future biological experiments.
A further advantage offered by the (Boc)2-creatine
amino intermediates was the fact that the product was
easily purified by preparative RP-HPLC before removing
the t-Boc group.
The presence of the t-Boc group greatly increases the
lipophilicity of the compound favoring the purification by
RP-HPLC with water and acetonitrile. This advantage is
particularly important when considering the difficulty in
analyzing, and hence purifying creatine and its derivatives
with chromatographic methods. The purification of creatine
and its derivatives without the t-Boc group involves using
salt buffers which, however, cause the final problem of
having to remove the salts formed (Dash and Sawhney
2002; Smith-Palmer 2002).
Conclusions
Creatine derivatives may have an important role for the
treatment of neurological diseases. Unfortunately, it is a
zwitterionic molecule which hardly reacts with other
compounds and it is also practically insoluble in organic
solvents. We solved these two problems by synthesizing a
particular kind of creatine with the guanidine group pro-
tected by two t-Boc groups. The (Boc)2-creatine was then
However, to be active the creatine derivatives must have
their guanidine groups free to interact with the creatine
123

832
P. Garbati et al.
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used to synthesize derivatives of creatine. In this paper, we
show the synthesis method of the (Boc)2-creatine and a
new simple method to synthesize creatine derivatives with
molecules having a reactive amino group. This method
allows obtaining the products synthesized in high yield and
with a purity greater than 95 %, due to the ease with which
they can be purified by RP-HPLC before removal of the
t-Boc groups. This method is especially suitable for syn-
thesis of amide derivatives, but does not exclude the ability
of (Boc)2-creatine to react with other nucleophilic groups.
Acknowledgments The authors would like to thank Dr. Marta
Bellotti for her valuable help in verifying (Boc)2-creatine stability.
Conflict of interest The authors declare that they have no conflict
of interest.
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