Sunteți pe pagina 1din 4

B I O PR O C E S S TECHNICAL

Carbohydrates and
Their Analysis, Part One
Carbohydrate Chemistry

Adriana E. Manzi

A
mammalian cell exists,
interacts, reproduces, and dies
as a result of intricate
biochemical and biophysical
mechanisms — many of which remain
unknown or not fully understood even
today. Technological advances have
allowed us to investigate the details of
organelles and cell membranes, of the
types of molecules synthesized within
cells, and their distribution and
trafficking and the effects they have
in their environment. Among the
main macromolecules that make up a
living cell, DNA and protein
structures were studied in detail long WWW.PHOTODISC.COM
before technologies enabled analysis of
the inherently more complex and less outer surface of cells — are in a THE BASIS FOR
predictable carbohydrate structures. position to modulate cell interactions, STRUCTURAL COMPLEXITY
Today, detailed analysis of complex making them relevant players in the Carbohydrates are substances that
carbohydrates remains challenging understanding of human health and satisfy the formula (CH 2O) n — as
because of the close similarity between disease. But training in carbohydrate well as many derived substances —
them and the very large number of chemistry, biochemistry, and biology and the term came about long before
possible molecular species involved. lags behind the routine teaching of detailed knowledge of the structural
It is widely recognized that courses related to DNA and protein features was available. From a
complex glycans — expressed on the sciences. Thus, the popularity of chemical standpoint, carbohydrates
carbohydrate studies declined from are polyhydroxyaldehydes (or
the beginning of the 20th century aldoses) or polyhydroxyketones (or
PRODUCT FOCUS: GLYCOPROTEINS when industrial applications of ketoses) and their derivatives (Figure
PROCESS FOCUS: ANALYTICAL polysaccharides were widespread. 1). The simplest ones are
While we ponder whether we are monosaccharides that can be linked
WHO SHOULD READ: QA/QC, PRODUCT actually living at the dawn of the era together to form oligosaccharides
DEVELOPMENT, PROCESS DEVELOPMENT, of “generic” biopharmaceuticals, and polysaccharides. The different
AND ANALYTICAL PERSONNEL perhaps we should take the time to number of carbon atoms in a
learn about these complex molecules. monosaccharide leads to the names
KEYWORDS: GLYCANS, CARBOHYDRATES, Their analysis is a critical component aldotriose, ketohexose, and so on.
OLIGOSACCHARIDES, PAT, COMPARABILITY in the demonstration of molecular Glyceraldehyde is the simplest
LEVEL: INTERMEDIATE comparability. aldose and has one asymmetric

54 BioProcess International FEBRUARY 2008


Figure 1: General formula for a
(chiral) carbon atom, therefore polyhydroxyaldehyde (aldose) and a
exhibiting optical isomerism. That polyhydroxyketone (ketose)
is, it can exist in the form of two ������������ �������������
nonsuperimposable mirror images Figure 2: D- and L- glyceraldehydes
called enantiomers (Figure 2). �������� ������
��� ���
Enantiomers have identical physical
properties except for the direction of ������������� ������� � � �� �
rotating the plane of polarized light
(–, left-hand or levorotatory; +,
������������ �����
right-hand or dextrorotatory). The ������ ������
pair also has identical chemical ��������������������������� ���������������� ����������������
properties except in relation to
optically reactive reagents. Figure 3: Aldohexoses of the D - series
The simplest ketose is
dihydroxyacetone, which contains no ��� ��� ���� ��� ��

asymmetric carbon atom. All other �� ��� � �� �� �� ��� � �� �� ��
monosaccharides contain at least one �� ��� �� ��� � �� �� � �� ��

��
chiral carbon atom. The total �
number of asymmetric centers equals �� ��� �� ��� �� ��� �� ��� ��

the number of carbon atoms in a �� ��� �� ��� �� ��� �� ��� ��

molecule minus the number of ��
�� ��� �� ��� �� ��� �� ���
end-chain carbon atoms for an
aldose (n – 2), and minus the ������ � ������� ������� �������
end-chain carbon atoms and the
��� ��� ��� ��� ��
nonasymmetric ketone carbon for a �
ketose (n – 3). As the number of �� ��� � �� �� �� ��� ��� � ��
carbon atoms in a chain increases, �� ��� �� ��� �� � ��� �

��
the number of possible stereoisomers �
also increases. The total possible �� �� ��� �� �� � ��� � ��

number of stereoisomers can be � ��� �� ��� �� ��� �� ��� ��

calculated as 2k , where k is the ��
�� ��� �� ��� �� ��� �� ���
number of asymmetric carbon
atoms. Therefore, a carbohydrate ������ ����� � ��������� � �������
with the formula C5 H10 O5 and
Figure 4: Aldopentoses of the D - series
three asymmetric carbon atoms
has eight possible stereoisomers �� ��� ���� ���� ��� � ��
� � �
(Figures 3 and 4). � ��� �� �� �� � ��� �� ��
�� � �� �
The carbon atoms in a � �
� �� ��� ���
monosaccharide are numbered �� � �� � �� � �� �� ��
� �
following the rules of organic ��

� �� � �� �� � �� �� � � �� � ��
chemistry nomenclature. Thus, � �
�� ��
the aldehyde group is C-1, and ��� �� � ��� �� � ��� �� � ��� �� �


the ketone group is C-2. The ������ � ��������� � �� ������ � �� ������


prefix D- (for dextrorotary) is �
added to each monosaccharide Figure 5: Representation of α and β anomers of D -glucopyranose as cyclic hemiacetal structures
that has the same configuration
as D-glyceraldehyde at the � � � �������� � ��������������������� �� � ��������

highest-numbered chiral carbon in its
� � �
structure (the CHOH group most � �� � ��� � �� �
distant from the aldehyde group and
�� �� � �� �� � �� ��
next to the last CH 2OH). Similarly, � �
the prefix L- (for levorotary) is added � �� �� � �� �� � �� �
to those monosaccharides with
�� �� �� � ��
the same configuration as �� � ��
L-glyceraldehyde at the highest- � �� �� � ��
numbered chiral carbon in its � �

structure. The D- configuration is �� � � � � �� � �� �� � �� �


predominant in nature.

56 BioProcess International FEBRUARY 2008


The direction of rotating the plane of the configuration of one chiral The formation of these cyclic
polarized light for a compound must carbon atom are called epimers. structures is favored when
be determined experimentally, and it In solution, monosaccharides exist chemically stable rings can be
is then indicated with the – or + sign mainly as cyclic hemiacetals or formed (five- or six-membered
(between parentheses) immediately hemiketals formed by condensation, rings). In an aldohexose, the
after the prefix D- or L-. Two having free hydroxyl groups within condensation reaction between the
monosaccharides that differ only in the same monosaccharide (Figure 5). aldehyde group at C-1 and the
hydroxyl group at C-5 originates a
Figure 6: Fischer representation and Haworth projection formula for α- and β- D -glucopyranose � six-membered ring called pyranose.
When the C-1 of an aldopentose
���� � ������������� reacts with the C-4 hydroxyl group,
��
�� �� a five-membered ring is formed

� �� � �� � �� � �� � �� � called furanose. The same
��
� �� � �� �� � aldopentose can originate a

� �� � �
� � ��
�� ��
�� �� ��
�� ��
� six-membered ring by reaction

�� �� �� �� � �� �� � of the C-1 aldehyde with the C-5
�� �
� �� �
�� �
�� �� � hydroxyl group. The C-2 of a

� �� � �� ketohexose can also react with the


C-5 hydroxyl group forming a
� furanose ring. This cyclization
����� � originates an additional asymmetric
���� � �������������� center at the original carbonyl group
�� �
called anomeric carbon.
��
�� � �� � �� � �� �
Because the hydroxyl groups
�� � �

�� �� �
attached to the anomeric carbon can
�� �� � �� �
�� �� � � adopt two possible spatial
�� � ��
�� �
�� �� ��
�� ��

orientations, that creates two
�� � � �� ��
�� � � �� � stereoisomers: alpha (α) and beta
� �� � �� �� �
�� �� � � (β). These two anomers (anomeric
isomers) are interconvertible and
��
��
exist in an equilibrium mixture in
����� ������������ solution. Interconversion occurs
������������������ �
������������������ through the open-chain structure
�������� with the reactive aldehyde or ketone
�������������� � group. The α and β forms in
� equilibrium rotate the plane of
Figure 7: Chair representations of α- D -mannopyranose plane-polarized light an equal
amount but in opposite directions, so
�����
����� the optical rotation rapidly changes

�� �� �� � �� (mutarotation) when any of the pure
forms is dissolved in water toward a
�� �� final equilibrium. In spite of the
�� ��
small proportions of reactive
alhedyde or ketone present in that
Figure 8: The disaccharide α-maltose in its preferred conformation (α anomer shown for the
reducing end) equilibrium, a sugar can participate
in chemical reactions of either cyclic

or open-chain form.
�����
� It is important to visualize the

�� differences between monosaccharide
structures. For this purpose, different

�� � conventions were developed. Linear
� �� ����� Fischer projection formulas are useful

� for understanding the relationship
� � between different common
� monosaccharides. Applying the same
��
convention to cyclic hemiacetals
� �� results, however, in an awkward
���������������������������������������� � �� representation of the ring structure

58 BioProcess International FEBRUARY 2008


that can be more realistically depicted LOOKING AHEAD
using a Haworth projection formula The next installment of this three-part
(Figure 6) or even more so using a article will address structural
chair conformation (Figure 7). characteristics of polysaccharides and
Conversion between Fischer and glycoconjugates. Part Three will focus
Haworth representations must follow on their analysis and its application in
a number of rules. For a D- bioprocess monitoring.
monosaccharide, any group directed
to the right in the Fischer projection ACKNOWLEDGMENTS
should be drawn downward in the The author thanks Dr. Nadine Ritter, Dr. Kalyan

critical review of this multipart manuscript. 


Anumula, and Dr. Panneer Selvam for their
Haworth projection; conversely, if a
group is written to the left in Fischer,
it should go upward in Haworth; the
terminal –CH 2OH group goes Adriana E. Manzi, PhD, is president of
upward in Haworth. For an L- Manzi & Associates, 6051 Scripps Street,
monosaccharide, the terminal San Diego, CA 92122; 1-858-554-0636;
–CH 2OH group goes downward in amanzi@san.rr.com.
the Haworth projection.
The preferred conformation of a
pyranose ring is the chair
conformation. Hydroxyl groups in
such a conformation are either axial
or equatorial. Two chairs in
equilibrium are called conformers. The
position of that equilibrium differs
from one monosaccharide to another
depending on the relative position of
the hydroxyl groups or other
substituents. The preferred
conformation has the lowest number
of bulky groups in axial positions.
When a condensation reaction
occurs between the anomeric
hydroxyl group of one
monosaccharide and any hydroxyl
group on another, the reaction
produces a disaccharide. The
covalent bond between the two
moieties is called glycosidic linkage,
which is said to be either α or β
based on the configuration of the
anomeric hydroxyl group involved
(Figure 8). Two disaccharides
constituted by the same two
monosaccharide units and differing
only in the position or configuration
of their glycosidic linkage may
have different conformations,
resulting in different physical
properties. Complex carbohydrates
combine the inherent structural
rigidity of ring structures with the
f lexibility of a glycosidic linkage and
thus can vary their conformation in
solution. Larger structures are
formed by subsequent glycosidic
linkages (trisaccharides,
tetrasaccharides, and so on).

60 BioProcess International FEBRUARY 2008

S-ar putea să vă placă și